WINTER 2023 | VOL. 37, ISSUE 4 | WWW.THE-SCIENTIST.

COM

RECENT ADVANCES IN
MODELING THE HUMAN
PLACENTA MAY INFORM
PLACENTAL DISORDERS
LIKE PREECLAMPSIA.
Revealing Immune Responses
with Adaptive Immune
Receptor Repertoire Profiling
Revealing Immune Responses with Adaptive
Immune Receptor Repertoire Profiling
BACKGROUND: Receptor Diversity TECHNOLOGY: What Is the DriverMapTM Adaptive Immune
T cell and B cell receptors (TCRs and BCRs) are key drivers Receptor (AIR) Repertoire Profiling Assay?
of adaptive immunity. TCR and BCR coding sequences are
The DriverMap human and mouse AIR-RNA profiling assay
arranged via a process known as V(D)J recombination. Within
uses targeted multiplex RT-PCR to profile all functional TCR
a given cell, variable (V), diversity (D), and joining (J) segments
and BCR isoforms (TRA, TRB, TRG, TRD, IGH, IGK, and IGL
are randomly selected from many variants and combined to
chains) in a single experiment while excluding non-functional
generate the full-length receptor. Scientists estimate that there
pseudogenes and open reading frames. A single-tube multiplex
are approximately 200 million T and B cell clones with distinct
RT-PCR followed by next-generation sequencing (NGS)
receptor sequence combinations present in human blood.
enables robust rapid profiling of human or mouse RNA, DNA,
or both. The simultaneous profiling of DNA and RNA enables
the identification of antigen-activated clonotypes to provide
insight into the immune response.
V-segments D-segments J-segments Constant region

1 D to J recombination

mRNA V (D) J C AAAAAAAA

UM
I

RevGSP
An

2 V to DJ recombination
ch
or

mRNA/GSP Hybridization
2

3 Transcription & splicing mRNA 5’ V (D) J C AAAAAAAA

UM
I

AAA cDNA 3’ V (D) J RevGSP

An
 nc
ho
r2
cDNA Synthesis from RevGSP

An
ch
or
1
(D) J C UMI
4 Translation & assembly RevGSP UMI Anchor 2
3’ V (D) J 3’

FwdGSP
Extension

P5
TCR and BCR Structure Anchor 1

(D) J C UMI 3’
The variable region of both BCRs and TCRs contain two
variable regions 3’ V (D) J RevGSP UMI
polypeptide chains: light and heavy. These chains each Anchor 2
P7
PCR from Anchor Primers
contain three complementarity-determining regions (CDR1,
NGS
CDR2, and CDR3). The V(D)J recombination junction is
located in CDR3, making it the most diverse of the three
CDRs and directly involving it in antigen recognition. CDR3 is
therefore the primary focus of much scientific investigation.
constant regions

transmembrane region

How Does the DriverMap AIR Profiling Assay Work?

The Power of Immunosequencing
Immunosequencing amplifies rearranged CDR sequences, and
when combined with high-throughput sequencing technology,
has the power to enumerate and quantify thousands of TCR
or BCR clonotype sequences simultaneously. Scientists
can use this information to characterize the abundance
and distribution of lymphocytes, as well as to track clonal
migration over time during situations such as disease
progression or immunologic responses.2
Isolate total RNA or genomic DNA from any type
of immune sample such as whole blood, PBMCs,
1 cancer biopsies, tissue samples, FFPE, or dried blood
microsamples.
Amplify AIR clonotypes either with the multiplex PCR-
targeting CDR3 variable and conservative regions of the
T- and B-cell receptors, or with the full-length assay that
2 captures CDR1, CDR2, and CDR3 regions.
Use NGS with to profile clonotypes present in each
3 sample. UMI-labeled primers enable quantitative analysis of
clonotype representation.

To Start With DNA or RNA?

AIR repertoire profiling can be done with both genomic DNA (gDNA) and RNA,
and each has its own pros and cons.
 RNA gDNA

Sensitivity 10-100 fold higher than DNA Relatively low

Functionality Only amplifies functional/expressed genes Amplifies many non-rearranged and non-functional sequences

Coverage Can identify Ig isotype Cannot identify Ig isotype

Quantitation Cannot assess T/B cell counts per clonotype Can assess T/B cell counts per clonotype

RESULTS: Adaptive immune repertoire Immunophenotyping

The DriverMap AIR assay facilitates simultaneous profiling of all CDR3 or (AIR) Profiling of T/B marker genes
full-length variable region TCR (TRA, TRB, TRD, TRG) and BCR (IGH, IGK,
IGL) clonotypes in a single test-tube reaction. The comprehensive analysis
of both TCR and BCR repertoires helps researchers better understand how both
arms of the adaptive immune system work synergistically.

Running both AIR-RNA and AIR-DNA assays on the same sample allows for
the identification of antigen-activated clonotypes by showing transcriptional
activation of TCR and BCR genes in immune responses.

DriverMap AIR assay repertoire profiling is sensitive and reproducible.

The assay reproducibly identifies and quantitates the most abundant 100-
500 clonotypes (with specific mRNA copy number exceeding 10) present in
RNA samples. It also detects 3,000-5,000 medium-abundancy clonotypes
reproducibly if replicates are used. Finally, the assay amplifies 50,000-500,000
low-abundancy clonotypes which are present at single-copy mRNA levels.

DriverMap AIR profiling and immunophenotyping assays complement each

other. The direct parallel profiling of AIR clonotypes and targeted expression Cd4Tn Cd4Te CD8Tn CD8Te TregN Cd4Tn Cd4Te CD8Tn CD8Te TregN

levels of 500 T/B cell-typing markers in sorted T cell fractions facilitates TCR
repertoire characterization in specific immune cell subsets, including CD8
naïve, CD8 effector, CD4 naïve, CD4 effector, and regulatory T cell fractions,
providing greater insight into immune samples.
Parallel profiling of TRB clonotypes and immune cell
phenotypes in FACS-sorted CD4 naïve, CD4 effector,
CD8 naïve, CD8 effector, and regulatory T cell fractions.

DriverMap AIR
250K
200K
mRNA-DriverMap™ AI
180K
200K 160K gDNA-ImmunoSEQ®

types
types

140K
 250K 120K
SMART-based 100K
100K 80K
60K
50K 40K

Number of Clon
Number of Clon
20K
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
PBMC1 PBMC2 PBMC1 PBMC2
Sample Number

The DriverMap AIR assay identifies 3x more clonotypes The DriverMap AIR assay detects 1.5-2x more clonotypes than conventional
than a conventional SMART®-based 5’-switch oligo TCR assay. gDNA-based assays.

Bulk analysis or single-cell sequencing for AIR repertoire assays?

The bulk AIR assay is the best strategy for cost-effective, quantitative immune receptor repertoire analysis of a large number
of biological samples with the goal of identifying antigen-activated clonotypes or disease/treatment-associated TCR/
BCR biomarkers. Single-cell analysis is the most informative approach, but also the most costly strategy for the phenotypic
characterization of cell encoding TCR/BCR clonotypes and receptor chain pairings identified through AIR bulk analysis.

Bulk analysis T/B cell fractions Single-cell analysis

Throughput High Medium Lower

Sample Cells (intact or lysed), tissues, fluids FACS-sorted cellular fraction Intact live cells
or single cells

Complexity and Cost Easy, inexpensive Modern, less expensive Harder, more expensive

Scaling Simpler Less complex More complex

Data Depth Cannot provide information on receptor Provides information on receptor Can link clonotype repertoire with
chain pairings; cannot link phenotypes with chain pairing for single-cell sorting; paired-chain information and cell
clonotypes links cell phenotypes with clonotypes phenotype

Immune receptor repertoire profiling is an important analytic tool for disease research in many areas,
including cancer,2 cell and organ transplantation,3 autoimmunity,4 and infectious pathologies such as
COVID-19.5 The DriverMap AIR repertoire profiling assay offers researchers the ability to characterize their
immune cell samples in a comprehensive and sensitive manner. The assays are available as ready-to-use kits
or as a service with a rapid one-month turnaround.
Immune receptort repertoire profiling.n
Your new superpower.

Introducing the DriverMapM

™
Adaptive Immune Receptor o
(AIR) Profiling Assay

Start with total RNA or DNA

• Comprehensive profiling ofn all 7 TCR/BCR isoforms
u
• Accurate detection of functional isoforms only, not pseudogenes or ORFs
s
• Robust results from blood, tissue, FFPE or any immune sample
• No specialized instrument required

Now available as a kit or as a service.

Learn more at cellecta.com/drivermap-air

Who we are
Cellecta Inc., a trusted provider of genomic products and services, has successfully
collaborated with the world’s leading pharma, biotech, government, and academic
institutions since 2006. Our recognized expertise in viral vector production, functional
screening, cell engineering and multiplex qRT-PCR has given rise to a portfolio of
offerings useful for loss-of-function and gain-of-function phenotypic screening, cell
barcoding, targeted RNA-Seq and adaptive immune receptor profiling, and more.

We help power your discovery efforts.

© 2023 Cellecta, Inc. 320 Logue Ave.
www.cellecta.com info@cellecta.com +1 650-938-3910 Mountain View, CA 94043 USA
Cellecta Inc. is a trusted provider of genomic products and services for drug target and
biomarker discovery. Since 2006, we have collaborated with the world’s leading pharma,
biotech, government, and academic institutions. We apply our extensive expertise in viral
vector production, functional screening, custom cell engineering and multiplex RT-qPCR
to provide a variety of products and services, including:

• CRISPR and RNAi pooled libraries and loss/gain-of-function screening services to

identify genetic pathways responsible for phenotypes and biological responses

• Cell barcode libraries and constructs for cell tracking and analysis of clonal
variations within cell populations

• Transcriptome profiling, TCR /BCR profiling and digital spatial profiling for
biomarker discovery

• Custom cell engineering projects for cell assay development, and more

From our headquarters in Mountain View, California, we work with researchers worldwide
to power their discoveries. Learn how to put our expertise to work for you at Cellecta.com

References:

1. S. Teraguchi et al., “Methods for sequence and structural analysis of B and T cell receptor repertoires,”
Comput Struct Biotechnol J, 18:2000-11, 2020.

2. I. Kirsch et al., “T-cell receptor profiling in cancer,” Mol Oncol, 9(10):2063-70, 2015.

3. A. Minervina et al., “T-cell receptor and B-cell receptor repertoire profiling in adaptive immunity,”
Transpl Int, 32(11):1111-23, 2019.

4. H. Katoh et al., “Immune repertoire profiling for disease pathobiology,” Pathol Int, 73(1):1-11, 2023.

5. P.C. Taylor et al., “Neutralizing monoclonal antibodies for treatment of COVID-19,” Nat Rev Immunol,
21:382-93, 2021.
Helpful Resources

Comprehensive Adaptive Immune Receptor Profiling for

All Immune Cell Types — flyer
Get an overview of the DriverMap AIR Profiling Assay for immune
repertoire profiling starting from RNA or DNA

DriverMap AIR Repertoire (AIRR) Profiling Tech Guide

Learn all about the AIRR profiling techniques, strategies for designing
experiments and key applications

T-cell and B-cell receptor repertoire profiling for biomarker

discovery — scientific poster
View an outline of the DriverMap AIR workflow and a rheumatoid
arthritis case study

Synthetic Spike-in Controls for Immune Repertoire Profiling

— flyer
Ensure accurate and reproducible immune repertoire profiling results
with this set of RNA controls

Imunophenotyping of T-Cell and B-cell Receptor Clonotypes for

Biomarker Discovery — video (18:40)

For more information and to access the links to these resources,

visit Cellecta.net/ts-air-resources
LIKE US ON
FACEBOOK
Did you know that more than
2 million people follow The Scientist
on Facebook? Like our page to see
the latest news, videos, infographics,
and more, right in your news feed.

facebook.com/TheScientistMagazine
 Contents
WINTER 2023 | WWW.THE-SCIENTIST.COM | VOL. 37, ISSUE 4 ON THE COVER: © SCIENCE PHOTO LIBRARY, MICROSCAPE

18 Features
24

© SHUTTERSTOCK.COM, SCIEPRO; MODIFIED FROM © ISTOCK.COM, ILYA LUKICHEV; © ISTOCK.COM, BAWANCH AND KUDRYAVTSEV PAVEL
32 38

18
Downsizing DNA
Some species remove up to 90%
of their genomes during develop-
ment, but why or how this hap-
pens is still a mystery.
BY APARNA NATHAN, PhD
24
The Ephemeral
Life of the Placenta
Recent advances in modeling the
human placenta, the least under-
stood organ, may inform placental
disorders like preeclampsia.
BY DANIELLE GERHARD, PhD
32
Unraveling the Mystery
of Zombie Genes
Digging into how and why
some genes are resurrected
after death sounds morbid,
but it has practical applications.
BY IRIS KULBATSKI, PhD
38
A Story of FIRE and Mice
Studying how microglia
control myelin growth and
prevent its degeneration helps
scientists better understand
and address neurodegenera-
tive diseases.
BY NIAMH MCNAMARA, PhD
AND VERONIQUE MIRON, PhD

2 T H E SC I EN T I ST | the-scientist.com
 Department Contents
FROM THE EDITOR
5 When Scientists Collaborate,
Science Progresses
Behind every successful scientist,
there is another scientist.
BY MEENAKSHI PRABHUNE, PhD
7 CROSSWORD
46 Tracking Down Innate Immune Cells
in Multiple Sclerosis
A novel PET tracer targeting a recep-
tor in myeloid cells can help monitor
disease progression in a mouse model
of multiple sclerosis.
BY MARIELLA BODEMEIER LOAYZA
CAREAGA, PhD

FOUNDATIONS
8 Coming Into the Fold: DNA Origami
In 2006, Paul Rothemund trans-
formed the field of DNA nano-
technology when he unveiled an inno-
vative approach for making shapes
and patterns from genetic material.
BY DANIELLE GERHARD, PhD
11 Serendipity, Happenstance, and Luck:
The Making of a Molecular Tool
The common fluorescent marker GFP
traveled a long road to take its popular
place in molecular biology today.
BY SHELBY BRADFORD, PhD
15 Cre-loxP: A Genetic Engineer’s
Swiss Army Knife
Standing at the cornerstone of genetic 53 Making Standards Exceptional
research, Cre-loxP recombination
serves as molecular scissors for
precisely manipulating the genome.
BY LAURA TRAN, PhD
UNIVERSITY OF ALABAMA, BIRMINGHAM; © ISTOCK.COM, MIRROR-IMAGES
PROFILE
48 A Microbial Link to Parkinson’s Disease
Haydeh Payami helped uncover the
genetic basis of Parkinson’s disease.
Now, she hopes to find new ways 48
to treat the disease by studying the
gut microbiome.
BY MARIELLA BODEMEIER LOAYZA
CAREAGA, PhD
50 A Journey With Metabolism, Parasites,
and Cancer
Piet Borst led stellar work on cell
organelles, trypanosomes, and cancer
drug resistance during the golden age
of biology.
BY LAURA TRAN, PhD
Samantha Maragh has taken on
the difficult challenge of standardizing
assays, data norms, and terminology in
the ever evolving genome editing field.

MEENAKSHI PRABHUNE, PHD

LITERATURE 59
42 Rebranding Mitochondria METHODS
As scientists realize the multifaceted 56 Whenever, Wherever: Taking DNA
role of mitochondria, some feel that Amplification Outside the Lab PUZZLE ON PAGE 7
the “powerhouse of the cell” analogy Recombinase polymerase amplification E A R L A N Y
66 65
D S E E T S
64

is out of date. lets researchers rapidly replicate DNA

R I A I M O A E D K C O C
62 63 61
O M E R I B O S E D T I D E
BY DANIELLE GERHARD, PhD in the clinic, in the field, or even in the 60
6 59 58
8 57 566 55
E T R I S R A T N O S
International Space Station. T U S P P A R A O L G I A
54
G
53

44 Biosensors for Colorectal Cancer BY HANNAH THOMASY, PhD O R S

1
51
O C T
52 9
49 0
50
A T E
48

Engineered bacteria sound the alarm 47

G U A A
44 45 46
6
A O W
43
3
S L E R
on a common oncogenic mutation. 59 Finding the One in a Million
42
O R A L E P S G E
41
W A
1
S T
0
40

Phage display revolutionized peptide

39 36
6 3
37 38
BY HANNAH THOMASY, PhD B A N G S A G A S E E R
35
3 34 33 32 31 29 30
screening methods and unlocked S S T I
28
H U M
26 2
27
V H
opportunities in protein discovery O N I R H O N D
25
2
M I T O C
23 24
2

and development.
I A S L E P G O S T O
22 21 20
L E N O T M E S L Y S O S O
BY SHELBY BRADFORD, PhD 19
1 17
7 188
O M E C N Y R I I N T O C
16 14 15
F I T E E N P S L A M B S
13 12 11 10 9 8 7 6 5 4 3 2 1

WINTER 2023 | T H E S C IE N T IST 3


MANAGEMENT
PRESIDENT
Bob Kafato
bobk@labx.com
EXECUTIVE VICE PRESIDENT
Rob D’Angelo
rdangelo@the-scientist.com
CHIEF FINANCIAL OFFICER
Chris Pauze
cpauze@labx.com
EDITORIAL
GROUP CONTENT DIRECTOR
Kristie Nybo, PhD
knybo@the-scientist.com
EDITOR IN CHIEF
Meenakshi Prabhune, PhD
mprabhune@the-scientist.com
ASSISTANT EDITORS
Danielle Gerhard, PhD
dgerhard@the-scientist.com
Mariella Bodemeier Loayza Careaga, PhD
mblcareaga@the-scientist.com
Laura Tran, PhD
ltran@the-scientist.com
Hannah Thomasy, PhD
hthomasy@the-scientist.com
Shelby Bradford, PhD
sbradford@the-scientist.com
ENGAGEMENT SPECIALIST
Melissa Kay
mkay@the-scientist.com
CREATIVE SERVICES
SENIOR SCIENCE EDITORS
Niki Spahich, PhD
nspahich@the-scientist.com
Nathan Ni, PhD
nni@the-scientist.com
ASSISTANT SCIENCE EDITORS
Iris Kulbatski, PhD
ikulbatski@the-scientist.com
Deanna MacNeil, PhD
dmacneil@the-scientist.com
Charlene Lancaster, PhD
clancaster@the-scientist.com
4 T H E SC I EN T I ST | the-scientist.com
1000 N West Street
Suite 1200
Wilmington, Delaware 19801
E-mail: info@the-scientist.com
DESIGN & PRODUCTION ADVERTISING, MARKETING, EDITORIAL ADVISORY BOARD
ART DIRECTOR & ADMINISTRATION James Allison, PhD
PRODUCTION MANAGER
SALES DIRECTOR University of Texas
Erin Lemieux Key Accounts MD Anderson Cancer Center
elemieux@the-scientist.com Ashley Haire
Jack Gilbert, PhD
ashleyh@the-scientist.com University of California,
SENIOR DESIGNER
San Diego
Ashleigh Campsall SENIOR ACCOUNT EXECUTIVES
acampsall@the-scientist.com Western US, Western Canada, ROW Joseph L. Graves, Jr., PhD
Karen Evans Joint School for Nanoscience
OPERATIONS and Nanoengineering
kevans@the-scientist.com
MANAGER
Erich Jarvis, PhD
Meaghan Brownley Midwest and Southeast US Rockefeller University
mbrownley@labx.com Anita Bell
Ellen Jorgensen, PhD
abell@the-scientist.com Biotech Without Borders
OPERATIONS COORDINATORS
Sarah Bond ACCOUNT EXECUTIVES Mary Claire King, PhD
sbond@the-scientist.com Northeastern US & Europe University of Washington
Jesse Silverman Elaine Mardis, PhD
Alan Collier jsilverman@the-scientist.com Nationwide Children’s Hospital
acollier@the-scientist.com
Northeastern US Joseph Takahashi, PhD
Jenna Short University of Texas
Addyson Chambers Southwestern Medical Center
jshort@the-scientist.com achambers@the-scientist.com
H. Steven Wiley, PhD
SALES OPERATIONS Pacific Northwest National
Laboratory
Amanda Purvis
apurvis@the-scientist.com
Mikaela Swietlinska
mswietlinska@the-scientist.com
CIRCULATION SPECIALIST
Matthew Gale
mgale@labx.com
SUBSCRIPTION RATES & SERVICES
In the United States & Canada individual subscriptions:
$39.95. Rest of the world: air cargo add $25.
For assistance with a new or existing subscription
please contact us at:
Phone: 847.513.6029
Fax: 847.291.4816
E-mail: thescientist@omeda.com
Mail: The Scientist, PO Box 2015, Skokie, Illinois 60076
POSTMASTER: Send address changes to The Scientist, PO Box 2015, Skokie, Illinois 60076. Canada
For institutional subscription rates and services, visit
Publications Agreement #40641071 The Scientist is indexed in Current Contents, Science Citation Index,
www.the-scientist.com/subscribe or
BasicBIOS IS, and other databases. Articles published in The Scientist reflect the views of their authors and
are not the official views of the publication, its editorial staff„, or its ownership. The Scientist is a registered e-mail institutions@the-scientist.com.
trademark of LabX Media Group Inc. The Scientist® (ISSN 0890-3670) is published quarterly.
REPRINTS
Advertising Office: The Scientist, 1000 N West Street, Suite 1200, Wilmington, Delaware, 19801 Contact Amanda Purvis at apurvis@the-scientist.com.
 FROM THE EDITOR

When Scientists Collaborate,

Science Progresses
Behind every successful scientist, there is another scientist.

BY MEENAKSHI PRABHUNE, PhD

W
hen I first learned about the
eukaryotic cell structure, I
marveled at the sophisti-
cated simplicity of how organelles syn-
ergistically work with one another to
ensure smooth cellular processes. On
the surface, the interplay of thousands
of proteins and the symphony of gene
modulations underlying a function seem
chaotic. Still, as we look deeper, impres-
sive patterns emerge, both within and
between cells and across different tissues.
A remarkable aspect of living systems is
their ability to communicate and collab-
orate for success. The gut finds a way to
contact the brain; the placenta comman-
deers nutrients for the fetus; and cells
selectively remove parts of their DNA for
better organism health.
The same fundamentals hold true
for scientific success. Anyone who has
attended a conference knows that the
crowds outside the presentation rooms
often surpass those inside. Scientific lead-
ers buzz around the venues to catch up
with their peers located across the globe
and to brainstorm future projects. Beyond
conferences, there is no dearth of trium-
phant stories where academics found
unique ways to strike up an alliance.
In today’s digital age of emails and
social media, global outreach does not
seem like a big deal, but collaboration
has been the hallmark of science for a
MODIFIED FROM © ISTOCK.COM, NIALL

long time, even when communication
channels were limited.1 For decades, sci-
entists have managed to transcend disci-
plines, geographical boundaries, or per-
sonal beliefs to share their knowledge
and contribute pieces towards solving
big-picture puzzles.
If we dissect the journey of any break-
through that drove a field forward, we
would no doubt find scientific collabora-
If we dissect the journey of any breakthrough that drove a
field forward, we would no doubt find scientific collaboration
at the helm driving success.
tion at the helm driving success. As you biology forward. Whether in the story of
peruse the articles in this issue, I invite you a federal agency employee who found her
to look out for those timely connections scientific ally to launch a genome editing
that propelled cell biology and molecular consortium, a US researcher’s invitation

WINTER 2023 | T H E S C IE N T IST 5

 to a Japanese researcher that led to GFP adorning heroes sprang into action and
as a molecular tool, a chance meeting their years of experience working together
during a seminar that led to significant globally paid off. Whether by sequencing Meenakshi Prabhune, PhD | Editor in Chief
progress in DNA origami technology, or samples globally to track COVID-19 vari- The Scientist
an interlab study that created new trans- ants, working together on vaccines, or
genic mice, you are bound to find a narra- developing scalable diagnostic tests, ded- References
tive that resonates with you. These often icated researchers kept each other abreast 1. Vermeulen N, et al. Understanding life together:
altruistic endeavors that help other sci- of their progress to help the world collec- a brief history of collaboration in biology.
entists take their research forward for the tively get through the pandemic.2,3,4 Endeavour. 2013 Sep;37(3):162-71.
2. Chen Z, et al. Global landscape of SARS-CoV-2
greater good of humanity truly embody The formats and forums may change
genomic surveillance and data sharing. Nat
the spirit of science. over the years with evolving needs and Genet. 2022;54:499–507.
This cooperative mentality was critical technologies, but I hope that the altruistic, 3. Druedahl LC, et al. Collaboration in times of crisis:
in helping everyone cope with the COVID- collaborative spirit of scientists remains A study on COVID-19 vaccine R&D partnerships.
19 pandemic. When the world was at a unchanged. If we do begin to lose it, we Vaccine. 2021 Oct 8;39(42):6291-6295.
4. Krijger PHL, et al. A public–private partnership
standstill with everyone helplessly trapped only need to look within our cells for inspi-
model for COVID-19 diagnostics. Nat Biotechnol.
at home, this special group of lab-coat- ration to bring it back. J 2021;39:1182–1184.

EDITORIAL

Scientific Our PhD-trained scientific

writers can sharpen your
manuscript's language and look.

Services
Our Scientific Services team can help you shape your
message and deliver it to the people who need to see it. COMMUNICATION
Our team can polish your
message and present it
Figure 3
Fi Hia
1 BD2 BD1 1098 to your audience.
Hsf
1 BD2 BD3 BD1 2414
BEFORE
AFTER
Figure 3. Do
transporters.
repetitive arc
consists off a Signal Peptide Neck/IsNeck domain
pared
d to Hia Hia
Trp ring domain GANG domain
daTAA A serve 1 BD2
KG connector domain
TTT domain
Y1head domain
B-barrel domain
BD1 1098
GRAPHICS
Hsf Our professional graphic
1 BD2 BD3 BD1 2414
designers will bring your
figures and schematics to life.

LEARN MORE
CROSSWORD

Cells and Organelles

ACROSS 1 2 3 4 5 6 7 8 9 10 11 12 13

1. Animal used in some preclinical research

14 15 16
5. Extremely common type of genetic
variation, for short 17 18 19
8. Consider it proper
14. Really loving 20 21 22

15. “Runaway Girl” actress Lili St. ___

23 24 25
16. Foundation grants for a lab, often
17. Organelle that breaks down 26 27 28
biological polymers
29 30 31 32 33 34 35
19. Illegally taken
20. Like slow city traffic 36 37 38 39
22. Mount Saint ___, fourth-highest peak
in North America 40 41 42

23. Organelle that generates cellular energy

43 44 45 46 47
26. Sound made by the plainfin midshipman

BY STELLA ZAWISTOWSKI
fish to attract mates 48 49 50 51 52
27. Old tape format
53 54
28. “___ the season to be jolly”
29. Wipe the data from 55 56 57 58 59 60
31. Succumb to gravity
32. Big ___ theory 61 62 63

36. Word that can follow “living” or “low”

64 65 66
37. Assimilation and ammonification in
the nitrogen cycle, for two
39. Taken by mouth
40. Like a salamander’s life history
4. Upward shove 35. Amorphous solid that’s highly useful
41. Subunit of an eon
5. “Move over!” to humans
42. Water: Spanish
6. Immature dragonfly, for example 37. Group that looks for life outside of Earth
43. Consumed
7. Response to “grazie” 38. Pay for dinner
44. Month in which Daylight Saving Time ends
8. Term of endearment for a sibling 43. Came to a consensus
47. Some Boolean operators
9. Goes in 44. Best possible conditions
48. Organelle that packages proteins
10. Bacteria with strains that can be symbiotic 45. Graphene’s makeup
into vesicles
with humans 46. Small musical group
53. Bat’s prey-detecting system
11. Large sheet of paper 48. Titular literary character who never shows up
54. Video game with pieces falling from above
12. “To explain...” 49. How some samples are shipped
55. After CRISPR-Cas9, say
13. “___ across the board!” 50. Potato pancake served at Hanukkah
57. Organelle that synthesizes
18. Apparatus with burners 51. Extreme danger
polypeptides and proteins
21. Coming up next 52. Process that may require sample
61. Tilted to one side
23. Artwork created by Montana State purification
62. Texter’s prelude to a hot take
University’s Bioglyphs project 53. Short moments?
63. Opera singer’s solo
24. Fully mature insect stage 56. Degree held by a dentist: Abbr.
64. Noble horses
25. Sites of archaeological interest 58. Magnetite, chromite, or cassiterite
65. Office computer setup: Abbr.
26. Cuts with a saw 59. Nowhere to be found, for short
66. 88 Earth days, for Mercury
30. Create suture 60. Body part that contains the vestibular nerve
31. Habitat for Pelagibacter ubique
DOWN 32. Snake that kills through constriction
1. Genre-bending artist ___ Nas X
33. Specialized jargon
2. One or more
34. Micronesian microstate known for
3. The Rockies, e.g. Answer key on page 3
its coral cliffs

WINTER 2 02 3 | T H E S C IE N T IST 7
 FOUNDATIONS

Coming Into the Fold: DNA Origami

In 2006, Paul Rothemund transformed the field of DNA nanotechnology when he unveiled
an innovative approach for making shapes and patterns from genetic material.

BY DANIELLE GERHARD, PhD

D
NA, the medium of life, is so deeply associated with the what I was talking about,” said Rothemund. He hit a dead end,
biochemical world that considering its nonbiological and following graduation in 1994, he joined a geobiology lab as
applications may seem far-fetched. However, for research- a technician.
ers in the 1980s and 1990s working in the fledgling field of DNA Later that year, Leonard Adleman, a computer scientist at
nanotechnology, it was more than a flight of fancy. the University of Southern California (USC), published a semi-
Nadrian “Ned” Seeman, the father of DNA nanotechnology nal paper in Science in which he used DNA to compute an algo-
and formerly a biochemist at New York University, first proposed rithm.5 “I simultaneously felt sort of scooped and validated that
the idea that DNA is not only a genetic material but also a con- there was something to the idea of encoding information in DNA
struction material in his seminal 1982 Journal of Theoretical Biol- molecules and doing computing,” said Rothemund.
ogy paper.1 A crystallographer by training, Seeman struggled to
crystalize proteins. He wanted to build DNA cages that were
strong enough to hold the protein in place long enough to take a
great picture. En route to tackling this problem, Seeman created
the field of DNA nanotechnology. “Ned had such a huge body of
literature that he’s sort of everyone’s go-to inspiration source,”
said Erik Winfree, a computer scientist and bioengineer at the
California Institute of Technology (Caltech).
In the decades that followed, grand ideas transformed into even
grander demonstrations as scientists repurposed nucleic acids to
store nonbiological information, such as all 154 of William Shake-
speare’s sonnets, and build microscopic robots for drug delivery.2,3

DNA computers
In the early 1990s as an undergraduate student at Caltech, Paul
Rothemund, now a computer researcher there, took a computer
science class that introduced him to the potential of DNA. The
professor discussed an idea from Charles Bennett, then a physi-
cist at IBM, that a computer built from DNA could simulate a
Turing machine wherein hypothetical enzymes could read the
Erik Winfree (left), Bernard Yurke (middle), and Paul Rothemund (right)
information stored in DNA and use that information to alter explore ways to expand the use of DNA.
DNA bases.4 “He said, ‘you know, someday, somebody who knows
something about computer science and biology or chemistry will
come up with a way to compute using DNA.’ At that moment, I Winfree recalled Rothemund’s class project on a similar topic
said, ‘Well, maybe I can do that,’” recalled Rothemund. and hunted him down to see if he wanted to attend the inaugural
He didn’t have to wait long. Rothemund had the opportunity one day workshop on DNA-based computers that would be held
to further explore the idea of building a molecular computer for at Princeton University in the spring of 1995. They scrounged
a project in another class. There, Rothemund met Winfree, then up the money to attend the conference. Winfree’s talk touched
a graduate student, and introduced him to Seeman’s work. on Seeman’s work on the self-assembly of DNA structures: the
As Rothemund neared the end of his studies, he wanted to spontaneous organization of molecules due to attractive forces.
continue working on the problem of building DNA computers, so After returning to his seat, Winfree felt Seeman tug on his
ERIK WINFREE

he shopped the project around. Computer science professors felt arm. Later that evening, Adleman, Seeman, Rothemund, and
ill-equipped to advise on the life science aspects of the project, Winfree huddled over a red and white checkered tablecloth in a
so he turned to life scientists. “The biology professors at Caltech pizza parlor and reflected on the day’s events. “That sort of seeded
and elsewhere told me that I was crazy, and they had no idea the next 25 years of my life,” said Rothemund. He joined Adle-

8 T H E SC I EN T I ST | the-scientist.com
Accessible
Gel Imaging
with a Cellphone

COVER PAGE
Accessible
Gel Imaging
with a Cellphone
Gel imaging enables scientists to see their samples in real time, but
capturing and analyzing bioimaging data can be a chore. Conventional
methods that rely on separate light boxes, manual-focusing cameras,
and computer interfaces turn a simple task into a tedious one, and limit
who can perform gel imaging experiments.
The UVP Gel Compact from Analytik Jena is an all-in-one, cellphone-driven UV
gel documentation system for fast and accessible gel imaging applications
applications.
When
W e p paired with
t thee VisionWorks
o o s Software
o ea and
d Software
o App, the
e App eUUVP Ge
Gel
Compact
p t allows
ll anyone
y tto use
eggel imaging,
g g, anytime
y e and
d anywhere.
y
All-In-One Gel
Documentation
High quality gel images allow researchers
to collect quantifiable information about
different types of experiments. Scientists
use gel imaging systems to see DNA and
protein in the form of bands on a gel,
or to count cells in a dish with precision.

1. Download the VisionWorks

App on your phone.
 Unique Features for
Stunning Gel Images
By directly connecting to the gel imager with
their own cellphones, users can visualize,
quantify, and analyze samples, making high
quality gel imaging easy in different settings,
from high school classrooms to academic and
industry research laboratories.

2. Connect to the UVP Gel

Compact via Bluetooth.
The Perfect Match for Education
Gel imaging can be an accessible experimental approach for novices and
experts alike. Beyond research, key features of the UVP Gel Compact and
VisionWorks Software make this powerful new technology the perfect match
for educational settings.

User-friendly interface

Software-controlled
lighting and filters

Automated features
(focus, capture, and save)

Cloud analysis and

simple report sharing

3. Select your settings

and start imaging!
 UVP GEL COMPACT
CELLPHONE-DRIVEN UV GEL DOCUMENTATION SYSTEM

PRODUCT DETAILS
CAMERA
The UVP Gel Compact imager is an autonomous, independent, and Unique feature of using your own cellphone to take images
cellphone-driven bioimaging system designed for the documentation and of your sample. A phone adapter/holder is included with
analysis for various types of samples which include but are not limited to each unit allowing for a range of phone sizes that work
seamlessly with the UVP Gel Compact.
DNA gels, protein gels, and colony plates, etc. The UVP Gel Compact allows
users to capture images and analyze them via their personal mobile device ILLUMINATION
equipped with Analytik Jena's VisionWorks Software App. The new
Overhead white and blue LED light
VisionWorks application was generated for easy gel capture and analysis
and allows for total control of the UVP Gel Compact darkroom.

The system features a 302 nm wavelength transilluminator, overhead

white and blue LEDs, and phone
ph h ld d
holder/adapter h can accommodate
that date
TRANSILLUMINATOR
The system features a 302 nm wavelength UV transilluminator.
most phone sizes on the market.
m k . Converter plates, UVP Visi-Blue LED Transilluminator, and a range
UV
of emission filters are available for purchase

Scan for more info.

APPLICATIONS
Designed for the documentation and analysis of various
types of samples which include but are not limited to DNA
gels, protein gels, and colony plates, etc.

SOFTWARE AND AUTOMATION

Connect to the darkroom via Bluetooth and operate on your
smartphone with our new VisionWorks® App (available for
CONTACT US: iOS and Android)
PHONE NUMBER: 909-946-3197
EMAIL ADDRESS: INFO@US.ANALYTIK-JENA.COM
WEBSITE: WWW.ANALYTIK-JENA.COM.COM
Analytik Jena is a leading provider of life science
products, including BioImaging, PCR, and Real-Time
PCR systems for academic, pharmaceutical and
biotechnology applications. Specializing in DNA/RNA
amplification and fluorescence and luminescence-based
imaging applications for proteomics, genomics and plant
imaging - our products are focused to offer customers
and users high quality and reproducible laboratory
results. A full line of Ultraviolet and laboratory products
for research based labs is also available.
UVP BioSpectrum Advanced
All-in-one fully automated imager

TAKE YOUR
IMAGING TO THE
NEXT LEVEL
UVP BIOSPECTRUM ADVANCED QUANTITATIVE IMAGING FOR
SYSTEM FEATURES WESTERN BLOTS AND MORE
Two ultra-cooled camera options to suit
different application needs paired with a fixed
low-light lens
Multiple illumination sources with overhead
longwave UV LEDs and white light, laser bars in
blue, green, red/NIR1, and violet (405 nm)
uniformity and coverage
Automated 6-position slide-out emission filter
tray with automatic filter type detection.
Motorized lift with precision stepper motor and
independent constant illumination
VisionWorks Analysis and Acquisition software
with sophisticated 21 CFR part 11 compliance
support with user management capabilities
In-Vivo imaging capabilities: Imaging for up to 10
mice, photon flux imaging calibration, narrow
band-laser excitation and narrow bandpass filters for
precise removal of background from the desired signal

info@us.analytik-jena.com
2066 W. 11th Street
Upland, CA 91786
909-946-3197
 man’s lab at USC later that year as a graduate student, while Win-
free ventured to the east coast to collaborate with Seeman at New
York University on the self-assembly of DNA crystals.6
Folding DNA
DNA computing captured the imaginations of many entering the
nascent field of DNA nanotechnology by demonstrating a non-
biological application for nucleic acids. However, by the turn of
the century, many researchers shifted their focus. “Many in the
field convinced ourselves that although this was intellectually
stimulating, this was not going to compete with electronic com-
puters,” said Winfree.
Rothemund focused his efforts on building nanostructures
using DNA or programmable approaches for DNA assembly.
Specifically, he considered how self-assembly processes could be
treated as algorithms and studied using computer science tools.
In 1993, Seeman detailed the construction of complex nano-
structures via the self-assembly of molecules.7 He was interested
in figuring out how to design molecules that self-assemble to form
parallel DNA helices where strands cross over and become part
of another double helical line, thus stitching together the helices.
Over the next decade, this inspired others in the field to innovate,
increasing the number of crossovers and helices.
Rothemund and Winfree’s paths crossed again in 2001. Win-
free returned to Caltech as a professor, and Rothemund joined his
lab as a postdoctoral researcher, where they continued their work
on algorithmic self-assembly of DNA.8
DNA is a versatile building block, but Rothemund described
DNA as optically, biochemically, and electronically dead com-
PAUL ROTHEMUND
For the first DNA origami experiments, Rothemund created one-third of a
square. By adding only one-third of the required staple strands, only one-
third of the square folded, resulting in rectangular shapes captured using
atomic force microscopy. The “ss” label denotes unfolded single-stranded
scaffold; “s,m” denotes a stable monomer; and “u,m” denotes an unstable
monomer. The scale bar is 100 nm.
pared to other molecules and materials like quantum dots, car-
bon nanotubes, or antibodies. “But what it can do is you can use
the information in DNA sequences to build structures, and then
you can use that structure to organize those other things,” said
Rothemund. Since the available methods for creating shapes out
of DNA were laborious and time consuming, Rothemund set out
to develop an easier approach. “And really, that was the idea for
DNA origami,” he said.
Rothemund’s DNA origami consisted of two main com-
ponents: a long, single-stranded piece of bacteriophage DNA,
which serves as the scaffold material, and a bunch of shorter
strands of oligonucleotides, or staple strands, that fix the struc-
ture in place.9 He fed his design into a computer program that
used principles of Watson-Crick base pairing to determine which
sequences were needed to instruct the scaffold strand to fold into
the desired shape or pattern. In his one-pot method, Rothemund
mixed the scaffold strand with the custom-made staple strands
and waited patiently as molecular self-assembly took shape.
Then, to confirm the structures, he used atomic force microscopy.
At the time, Winfree granted his lab members the freedom
to independently explore their interests. “There was a period in
2005 when I didn’t see [Rothemund] around very much,” said
Winfree. Eventually, Rothemund re-emerged with something to
share. “He showed me his results on the DNA origami, and to
tell you the truth, my first reaction was ‘Blech! Where’s the algo-
rithm? Where’s computer science?’” Uninterested in collaborat-
ing on the project, Winfree suggested that Rothemund publish it
himself, and so he did.
Winfree eventually came around to DNA origami. “It’s fan-
tastic. It’s revolutionized the field,” said Winfree. “[Rothemund’s]
demonstration was so elegant and thorough that it really opened
people’s eyes to how powerful the idea was.”
DNA origami’s spiritual successors
DNA origami isn’t the only approach for DNA assembly, but it’s
robust and relatively easy.10 “It’s the ability to create a geomet-
rically structured testbed for your experiment with each mole-
cule in the right place, and now, hundreds to thousands of mol-
ecules,” said Winfree. “That was unprecedented. That opened
up an ability to do experiments in all sorts of fields that people
previously couldn’t do.” For Winfree, that was putting short DNA
sequences in the correct order to trigger self-assembly. For oth-
ers, it was putting enzymes in the right order to produce a cas-
cade of enzyme reactions or quantum dots in a particular orga-
nization to control optics.
“One of the things that DNA origami has been able to do
since that paper was published is not something that’s widely
used in everybody’s cell phone or something like that, but it’s
a research tool for other things,” said Rothemund. He noted
that these custom instruments for biology allow researchers to
begin asking questions about proteins or other biomolecules
and even translate these ideas into therapeutics and molecu-
lar diagnostics.11,12
WINTER 2 02 3 | T H E S C IE N T IST 9
 FOUNDATIONS
Paul Rothemund created shapes, like smiley faces, and patterns, such as
DNA lettering, using his DNA origami method.
William Shih, a biochemist at Harvard Medical School,
employs principles of DNA origami in his quest to build
nanoscale objects, including molecular robots. In 2005, at a
conference in Albany, Shih learned what Rothemund had been
up to. “I saw his talk, and my jaw dropped because these images
that he produced—nobody had seen anything like this,” said
Shih. He recalled a particularly memorable image of DNA ori-
gami with “Ned” patterned on top in homage to Seeman. Still
mesmerized by Rothemund’s creations, Shih returned to his lab,
scrapped what he was doing, and pivoted to Rothemund-style
DNA origami.
“To me, what’s most special about DNA origami is that you
have an excess of building blocks that do absolutely nothing
except when they see a copy of this master controller scaffold
strand,” said Shih. Ten copies of the scaffold strand produce 10
DNA origami structures if there are sufficient staple strands.
Around the same time that Rothemund tinkered with DNA
at Caltech, Shih worked as a postdoctoral researcher down the
road at the Scripps Research Institute. In 2004, he published a
paper in Nature demonstrating the construction of a nanoscale
octahedron.13 However, Shih’s DNA folding approach required the
assembly of a substantial number of short, cloneable DNA struc-
1 0 T H E SC I EN T I ST | the-scientist.com
tures, with the idea that they could potentially enter bacteria and
replicate. Shih noted that without a master scaffold strand, the
construction is harder to control. “It’s kind of like herding cats,”
said Shih.
Recently, Shih’s research group has been busy develop-
ing what he referred to as the spiritual successors of DNA ori-
gami. The scale of DNA origami structures is limited by the
length of the scaffold strands, which is typically on the order of
10,000 nucleotides. Therefore, building anything bigger than
that requires forgoing the leading scaffold strand. To address
this, Shih and his team recently developed crisscross DNA ori-
gami whereby a controller molecule on the scale of a single
DNA origami directs the construction of a larger 1,000 DNA
origami structure.14 “They’re more like well-trained dogs than
cats,” said Shih.
Shih hopes that these advances will facilitate the construction
of bigger nanorobots with the size and complexity of a bacterial
or mammalian cell. He views this as a complementary technology
advancement to the broader field of synthetic biology where scien-
tists modify the genomes of living cells. “But they’re still living cells,”
said Shih. They’re still surrounded by a membrane; they still have
metabolism; and they still do DNA replication. “It’s important, tech-
nologically, to have alternate schemes that maybe don’t have a mem-
brane, that are not beholden to the normal process of DNA replica-
tion and translation, that maybe can be deployed in environments
that are hostile to living cells,” said Shih. J
References
1. Seeman NC. Nucleic acid junctions and lattices. J Theor Biol. 1982;99(2):237-247.
2. Goldman N, et al. Towards practical, high-capacity, low-maintenance information
storage in synthesized DNA. Nature. 2013;494:77-80.
3. Douglas SM, et al. A logic-gated nanorobot for targeted transport of molecular
payloads. Science. 2012;335(6070):831-834.
4. Bennett CH. Logical reversibility of computation. IBM J Res Develop.
1973;17(6):525-532.
5. Adleman LM. Molecular computation of solutions to combinatorial problems.
Science. 1994;266(5187):1021-1024.
6. Winfree E, et al. Design and self-assembly of two-dimensional DNA crystals.
Nature. 1998;394(6693):539-544.
7. Fu TJ, Seeman NC. DNA double-crossover molecules. Biochemistry.
1993;32(13):3211-3220.
8. Rothemund PWK, et al. Algorithmic self-assembly of DNA Sierpinski triangles.
PLoS Biol. 2004;2(12):e424.
9. Rothemund PWK. Folding DNA to create nanoscale shapes and patterns. Nature.
2006;440:297-302.
10. LaBean TH. Reminiscences from the trenches: The early years of DNA nanotech.
In: Jonoska N, Winfree E, eds. Visions of DNA Nanotechnology at 40 for the Next
40. Natural Computing Series. Springer, Singapore; 2023:55-67.
11. Andersen ES, et al. Self-assembly of a nanoscale DNA box with a controllable lid.
Nature. 2009;459:73-76.
12. Ochmann SE, et al. Optical nanoantenna for single molecule-based detection
PAUL ROTHEMUND
of Zika virus nucleic acids without molecular multiplication. Anal Chem.
2017;89(23):13000-13007.
13. Shih WM, et al. A 1.7-kilobase single-stranded DNA that folds into a nanoscale
octahedron. Nature. 2004;427:618-621.
14. Wintersinger CM, et al. Multi-micron crisscross structures grown from DNA-
origami slats. Nat Nanotechnol. 2023;18(3):281-289.
 FOUNDATIONS
Serendipity, Happenstance, and Luck:
The Making of a Molecular Tool
The common fluorescent marker GFP traveled a long road
to take its popular place in molecular biology today.
BY SHELBY BRADFORD, PhD
F
ollowing chemist and marine biologist Osamu Shimomu-
ra’s success in determining the structure of luciferin from a
crustacean at the Nagoya University, Frank Johnson, then
a biologist at Princeton University, invited him to study lumines-
cence in his lab. When Shimomura arrived at Princeton Univer-
sity in 1960, he had no ambitions for novel proteins and their
applications.
B. WEINSTEIN, NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT, NATIONAL INSTITUTES OF HEALTH VIA FLICKR.COM
Johnson was interested in in the blue light produced by a spe-
cies of jellyfish called Aequorea aequorea (now known as Aequo-
rea victoria), Shimomura began looking into the phenomenon. At
the time, scientists believed that all luminescence involved lucif-
erin and luciferase. However, the duo did not purify any luciferin-
related proteins from the jellyfish. This led Shimomura to believe
that the blue light must come from another protein, but he strug-
gled to isolate it.
A new way to luminesce
Shimomura attempted to isolate this mystery protein under var-
ious conditions. He finally found that he could inactivate the
luminescence with acid and obtain cell-free extract from the jel-
lyfish tissues. Satisfied with this finding, he dumped the used
samples down the sink. To his surprise, they glowed bright blue.
He reasoned that the substrate for his mystery luminescent pro-
tein must be in the sink, and that it certainly wasn’t luciferase.
The aquarium in the lab drained into the sink, so Shimomura
got to work testing the components in it to see if they elicited
blue light from his samples. Calcium turned out to be the culprit.
By removing calcium from the samples, Shimomura and
Johnson isolated the protein responsible for the blue light, which
they named aequorin.1 This was the first example of a light-acti-
vated protein, which they called a photoprotein.
I wanted to have a tool like this. I thought this
would be wonderful.
—Martin Chalfie, Columbia University
Today, we call them fluorescent proteins. Alongside aequorin,
Shimomura and Johnson also purified a small amount of another
protein that glowed green instead of blue. They called it “green
protein,” but at the time, they could not purify sufficient amounts
to study it further. While collecting and saving these samples for
future analysis, they continued exploring aequorin to character-
ize its structure and chemistry.
Fluorescent proteins enable researchers to study
biological processes in living organisms, as shown
in this zebrafish.
WINTER 2 02 3 | T H E S C IE N T IST 1 1
 FOUNDATIONS
Across the country in the mid-1960s, James Morin, a field
biologist who is now a professor emeritus at Cornell University,
became fascinated by bioluminescence. “It was amazing to me
that so many organisms that didn’t really have the capacities for
detecting luminescence would be luminescent,” he said.
This curiosity led him to the hydrozoan Obelia geniculata
when he began his graduate studies at Harvard University. He
noticed that the luminescence he saw from these animals in the
ocean was green, whereas in his prepared samples, it was blue.
“That indicated there must be some kind of coupling,” Morin said.
It turned out that O. geniculata, like Shimomura’s and John-
son’s A. aequorea, used a calcium-activated protein that emitted
blue light. This light activated a second protein that glowed green.
Morin published his findings from O. geniculata and coined the
term “green fluorescent protein” (GFP) for the first time in 1971.2
Shimomura confirmed that the same events happened in his jel-
lyfish in 1974.3
Shimomura continued to study GFP and identified its chro-
mophore in 1979.4 Ultimately, though, he returned to study-
ing bioluminescence. Morin also was more interested in the
function of luminescence and the nervous system of the sim-
ple marine species that produced it. “We were just interested in
the mechanisms of how that whole system worked,” Morin said.
“I’ve never been an applications biologist. I’ve been a discovery
kind of biologist.”
For the next two decades, GFP remained just an interesting
luminescent protein found in some species of marine animals.
Not just a jellyfish thing
In the 1970s, Morin, then at the University of California, Los
Angeles, took Paul Brehm on as a graduate student and intro-
duced him to Obelia and the field of bioluminescence. As a
biologist at Oregon Health and Science University, Brehm con-
tinued studying bioluminescence in Obelia as an associate pro-
fessor. In 1989, he presented his work at a seminar at Columbia
University; Martin Chalfie, a geneticist at Columbia University
who had never heard of GFP before, happened to attend. After
Brehm’s talk, Chalfie started tracking down GFP scientists on
the phone. “I was looking for where genes were expressed,”
Chalfie said. “I wanted to have a tool like this. I thought this
would be wonderful.”
In 1989, scientists studied genes of interest in cells using newly
developed technologies. “But in all of these instances, whether it’s
antibodies, or in situ hybridization, or using a reporter like beta
galactosidase, one needed to really prepare the samples. That
meant first they were fixed,” Chalfie said. “As a result, one had
really a snapshot in time.” There was no way to visualize what
genes were active in a living organism.
Chalfie thought that if GFP could be taken out of these
marine organisms and inserted into other cells, it could act as a
reporter in a living animal. However, nobody knew if this would
be possible. GFP had a cyclized portion in its structure, and
many people thought that such a complex chromophore struc-
1 2 T H E SC I EN T I ST | the-scientist.com
ture would need the help of other enzymes in the jellyfish to
fold correctly.
Chalfie’s phone calls eventually led him to Douglas Prasher,
a molecular biologist who worked at the Woods Hole Oceano-
graphic Institute. Prasher cloned the aequorin genes in 1985 and
began working on cloning the gene for GFP.5 The two struck up
a collaboration. In 1992, Prasher published his paper describing
the GFP cDNA clone, which he sent to Chalfie and his graduate
student, Ghia Euskirchen.6
With the clone in hand, the two had a decision to make.
Since Prasher cut the GFP gene out of the jellyfish genome with
restriction digestion enzymes, the clone had extra DNA seg-
ments that bookended the GFP gene. If Chalfie and Euskirchen
wanted only the GFP DNA, they could amplify just the GFP
gene by PCR, but that risked introducing mutations since PCR
was error-prone at the time. Alternatively, they could trans-
form the whole clone into E. coli, which would generate high-
fidelity DNA but retain the extra jellyfish sequence segments.
Because of this risk, most groups opted for transforming the
whole clone. “I know at least three groups that did that, and it
did not work,” Chalfie said. “We did something different.”
Taking a risk on error-prone PCR, Euskirchen amplified only
the GFP coding sequence and inserted it into an expression vec-
tor to transform back into E. coli. When Euskirchen imaged her
bacteria using a fluorescent microscope, she saw green glowing
back at her.
“That immediately said there is nothing else needed from the
jellyfish,” Chalfie said.
The jellyfish version of GFP just
crashed out in E. coli.
—Raphael Valdivia, Duke University
While this was a huge milestone in molecular biology, Chal-
fie believes that he owes some of his success to other contribut-
ing factors. “It’s not that I was the first person in the world who
ever thought about this,” Chalfie said. “It was a combination of
being at the right place at the right time, having the right people
in the lab and the right expertise to be able to try it, and then a
smidgen of luck.”
With this success, Chalfie and his team tested the initial goal
of inserting the isolated GFP into the touch-sensing cells of Cae-
norhabditis elegans. With positive data in hand, the group pub-
lished the first manuscript with GFP as a reporter in a nonma-
rine animal.7
An elegant measurement
Once the word was out that GFP could be put into other model
organisms, the protein found itself visiting labs across the coun-
 try, quite literally in the case of Raphael Valdivia, who is currently
a molecular geneticist at Duke University and who was a gradu-
ate student in Stanley Falkow’s lab at Stanford University in the
mid-1990s.
“This was when I had to take my qualifying exams to get into
PhD candidacy, and so I had to propose a project outside of my
thesis project,” said Valdivia. “I proposed to use GFP as a reporter
for gene expression in bacteria.”
Valdivia got the clone from Prasher and amplified the GFP
coding sequence to reinsert it into an expression vector, simi-
lar to Chalfie’s experiments, and then transformed it into E. coli.
“But when we expressed it, it did not work very well,” Valdivia
explained. Although the bacteria were green, the GFP took a long
time to fold and become fluorescent. “The jellyfish version of GFP
just crashed out in E. coli,” he said.
© ISTOCK.COM, AIMINTANG
Cells express GFP after viral transfection or insertion into the genome.
Valdivia wondered if he could tweak GFP to brighten its flu-
orescence. Working alongside Brendan Cormack, he created
mutants of GFP that targeted the protein’s chromophore.8 They
had their brighter mutants within a week.
“We didn’t do it with an idea of doing big technological break-
throughs or screens in general. We just needed something that
worked better in pathogens so we could screen,” Valdivia said.
How do you make a really elegant
measurement that’s biologically relevant
and learn things with it?
—Geoffrey Baird, University of Washinton
Valdivia eventually gave GFP to the spouse of a postdoctoral fel-
low in Falkow’s lab who worked at ClonTech, which is now Takara
Bio USA, Inc. Through this spurious connection, ClonTech opti-
mized GFP for expression in mammalian cells, licensed, and com-
mercialized as enhanced GFP, or eGFP in 1996.
While many groups worked with GFP to change its stability
or activity, Roger Tsien, a chemist at the University of California,
San Diego made possibly the most striking contributions. With
a background in dye chemistry, Tsien saw the value a fluores-
cent protein could offer biological research. According to Geof-
frey Baird, who was a graduate student in Tsien’s lab and is now
a clinical pathologist at the University of Washington, his mentor
had a fundamental interest that drove his research. “How do you
make a really elegant measurement that’s biologically relevant
and learn things with it?” Baird asked.
Tsien’s goal was to use GFP in fluorescence resonance
energy transfer (FRET) to monitor calcium signaling.
Although his team successfully generated a few mutants
that gave rise to altered colors of GFP, Tsien anticipated that
solving the structure of GFP would be important for more
modifications. 9-11
“Because it was only really with the structure that one could
then sort of say, well, hey, if we made this mutation here, we
might be able to make it a much different color,” Baird explained.
Tsien’s group solved this structure with help from James
Remington’s group at the University of Oregon in 1996.12 Within
a week, a second group published their finding of the struc-
ture, and the two closely aligned.13 With this information, Tsien’s
group got to work exploring what was possible with GFP. “I basi-
cally got into the business of making mutations,” Baird said.
Baird considers his most important contribution to have come
from an accident. He was trying to create brighter GFP clones,
and one day, he saw one that didn’t get dimmer after acid expo-
sure. After he sequenced it, Baird realized that he had made a mis-
take. “This thing shouldn’t be fluorescent at all,” he said.
WINTER 2 02 3 | T H E S C IE N T IST 1 3
 FOUNDATIONS
Researchers inserted a GFP tag to visualize a transcription factor for a
motor neuron in mouse embryonic stem cells and used a red-fluorescing
antibody against a marker for neurons to identify these cells.
Instead of a single point mutation, he had accidentally inserted
six amino acids into the sequence. But this accident showed that it
was possible to distort GFP. Baird informed Tsien of his mistake.
“He basically had all of the ideas for what you would do to exploit
a serendipitous finding,” Baird said.
Ultimately, the team found that breaking GFP and rejoining
it in a variety of ways through a process called circular permu-
tation created proteins with applications not only in FRET, but
also for studying protein interactions just by observing if a split
GFP molecule became active again.14 As Tsien had hoped and
anticipated, GFP was a very useful measuring stick.
Revolutionizing biology
From an obscure protein that wasn’t even abundant enough to
study, scientists have now published more than 40,000 papers
that reference green fluorescent protein. Today, GFP tags pro-
teins to view them inside of cells under microscopes and to iso-
late cells of interest using flow cytometry and fluorescence-
activated cell sorting.15 It has confirmed physical connections
between proteins and determined if a protein is made in the first
place. GFP has found itself in viruses, yeast, plants, mice, rats,
rabbits, pigs, and even nonhuman primates.16-23
1 4 T H E SC I EN T I ST | the-scientist.com
“It revolutionized cell biology and molecular biology,” said Baird.
Shimomura, Chalfie, and Tsien received the Nobel Prize in
2008 for their work on GFP.
“It’s been useful in the way I thought,” Chalfie said, reflecting
on GFP’s legacy. “But even more so, it’s been useful in ways that I
would have never imagined.” J
References
1. Shimomura O, et al. Extraction, purification, and properties of Aequorin, a
bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell
Physiol. 1962;59(3):223-239
2. Morin JG & Hastings JW. Energy transfer in a bioluminescent system. J Cell
Physiol. 1971;77(3):313-318
3. Morise H, et al. Intermolecular energy transfer in the bioluminescent system of
Aequorea. Biochemistry. 1974;13(12):2656-2662
4. Shimomura O. Structure of the chromophore of Aequorea green fluorescent
protein. FEBS Lett. 1979;104(2):220-222
5. Prasher D, et al. Cloning and expression of the cDNA coding for aequorin,
a bioluminescent calcium-binding protein. Biochem Biophys Res Commun.
1985;126(3):1259-1268
6. Prasher DC, et al. Primary structure of the Aequorea Victoria green-fluorescent
protein. Gene. 1992;111(2):229-233
7. Chalfie M, et al. Green fluorescent protein as a marker for gene expression.
Science. 1994;263(5148):802-805
8. Cormack BP, et al. FACS-optimized mutants of the green fluorescent protein
(GFP). Gene. 1996;173(1)33-38
9. Heim R, et al. Wavelength mutations and posttranslational autoxidation of
green fluorescent protein. Proc Natl Acad Sci. 1994;91(26):12501-12504
10. Heim R & Tsien RY. Engineering green fluorescent protein for improved brightness,
longer wavelengths, and fluorescent resonance energy transfer. Curr Biol.
T. MACFARLAN, NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT, NIH VIA FLICKR
1996;6(2):178-182
11. Sekar RB & Periasamy A. Fluorescence resonance energy transfer (FRET)
microscopy imaging of live cell protein localizations. J Cell Biol. 2003;
160(5):629-633
12. Ormö M, et al. Crystal structure of the Aequorea victoria green fluorescent protein.
Science. 1996;273(5280):1392-1395
13. Yang F, et al. The molecular structure of green fluorescent protein. Nat Biotechnol.
1996;14:1246-1251
14. Baird GS, et al. Circular permutation and receptor insertion within green
fluorescent proteins. Proc Natl Acad Sci. 1999;96(20):11241-11246
15. Chudakov DM, et al. Fluorescent Proteins and Their Applications in Imaging Living
Cells and Tissues. Physiol Rev. 2010;90(3):1103-1163
16. Oparka KJ, et al. Using GFP to study virus invasion and spread in plant tissues.
Nature. 1997;388:401-402
17. Li J, et al. Green fluorescent protein in Saccharomyces cerevisiae: real-time
studies of the GAL1 promoter. Biotechnol Bioeng. 2000;70(2):187-196
18. Köhler RH. GFP for in vivo imaging of subcellular structures in plant cells. Trends
Plant Sci. 1998;3(8):317-320
19. Yang M, et al. Visualizing gene expression by whole-body fluorescence imaging. Proc
Natl Acad Sci. 2000;97(22)12278-12282
20. Hakamata Y, et al. Green fluorescent protein-transgenic rat: a tool for
organ transplantation research. Biochem Biophy Res Commun.
2001;286(4):779-785
21. Takahashi R, et al. Establishment and characterization of CAG/EGFP transgenic
rabbit line. Transgenic Res. 2007;16:115-120
22. Brunetti D, et al. Transgene expression of green fluorescent protein and germ line
transmission in cloned pigs derived from in vitro transfected adult fibroblasts.
Cloning Stem Cells. 2008;10(4):409-420
23. Kurre P, et al. Kinetics of fluorescence expression in nonhuman primates
transplanted with GFP retrovirus-modified CD34 cells. Mol Ther.
2002;6(1):83-90
FOUNDATIONS
Cre-loxP: A Genetic Engineer’s
Swiss Army Knife
Standing at the cornerstone of genetic research, Cre-loxP recombination
serves as molecular scissors for precisely manipulating the genome.
BY LAURA TRAN, PhD
W
hen Nat Sternberg, a molecular biologist at the Fred-
erick Cancer Research Center, heard about bacterio-
phage P1, he was intrigued. Having previously worked
on the head proteins of h phage, Sternberg had an appetite for
studying phage-host relationships. P1 was largely unexplored, but
Sternberg was up for the challenge.
Like h phage, P1 infected Escherichia coli and entered the lyso-
genic cycle. In this stage, the bacteriophage genome integrated itself
into the host cell’s genome to achieve replication without destroy-
ing the host cell. During lysogeny, the h phage genome incorporated
into the host DNA, but the P1 phage displayed a unique feature:
It existed as an independent plasmid. P1 seemed to be a suitable
model for studying plasmids, so Sternberg set off to construct a P1
library in a h vector to study its genes.
He focused on studying site-specific recombination of P1. P1
phage particles were cyclic, and researchers expected recombination
to produce a circular map. However, Sternberg noted an unexpected
characteristic of P1’s recombination map. It was linear.
Sternberg pondered about the underlying mechanism; he sus-
pected that there must be a genetic hotspot for recombination
located at the terminal ends of the linear genome to facilitate
plasmid cyclization.1
Discovery of the Cre-loxP sandwich
Consistent with his hypothesis, Sternberg’s experiments revealed a
small fragment of P1 DNA at the end of the genetic map that was
likely responsible for site-specific recombinase. He named this locus
of crossover in P1, or a loxP recognition site. On further investiga-
tion, Sternberg performed deletion mutagenesis studies to iden-
tify another necessary component for recombination events, a P1
gene product he named Cre (an anagram for recombination).2 Cre
recombined target sequences at two loxP recognition sites.
He referred to the DNA sequences flanked by loxP sites as
“floxed.” The products of Cre-mediated recombination depend
on the orientation of the loxP sites. Two loxP sites oriented in
the same direction will excise DNA as a circular loop, while loxP
sites aligned in opposite directions will invert the DNA sequence
between them. This Cre-loxP sandwich in the P1 bacteriophage
laid the foundation for precise genetic manipulation.
When Sternberg moved to DuPont in 1984, a member of his
lab who followed him to DuPont, Brian Sauer, continued the proj-
ect. The biochemistry of the system was simple, but Sauer hoped
that Cre’s prokaryotic origins would not hinder its ability to effec-
tively work in eukaryotic cells. On testing loxP sites in yeast chro-
mosomes, Sauer was thrilled to see that Cre recombinase readily
recognized loxP sites and actively transported into the eukaryotic
nucleus.3 He later showed that Cre-loxP functioned efficiently in
mammalian cell lines.4
His next question was whether this system could be imple-
mented to target genes in the germline to obtain strains of geneti-
cally modified animals.
The explosive potential of transgenic mice
In the late 1980s, Nobel laureates Mario Capecchi at the Uni-
versity of Utah, Oliver Smithies at the University Wisconsin in
Madison, and Sir Martin Evans at the University of Cambridge,
pioneered methods for gene targeting in mouse embryo-derived
stem (ES) cells and homologous recombination as a mechanism
for manipulating genes in the mouse genome.5-7 This work dem-
onstrated the feasibility of making specific mutations in ES cells
and obtaining genetically modified knockout mice. This series of
breakthrough studies catapulted transgenic mice into the spot-
light and inspired researchers to follow suit.
Klaus Rajewsky, an immunologist from the University of
Cologne, took a keen interest in the development of the immune
system, especially that of B cells. He closely followed the work on ES
cells and applied it to develop one of the earliest knockout models of
the immune system by rendering mice deficient of B cells.8
However, the technology had its limitations. Rajewsky wanted
to study the function of DNA polymerase ` (pol`), but traditional
knockouts were not a viable option. Mice needed the gene early in
development, and eliminating this gene was fatal for them.
In addition to targeting a gene of interest, geneticists needed
to incorporate a selectable marker gene to find correctly recom-
bined cells. Rajewsky used the resistance gene neomycin in his
experiments, but he encountered an unexpected roadblock.
“Another problem that arose in these early knockout experi-
ments was the selection marker gene neomycin, which was still
in the locus and disturbed the phenotypes,” recalled Rajewsky.
“I, along with many others, thought about how this could be cor-
rected. Then, we came across Cre recombinase systems.”9 When
he read about this system, Rajewsky eagerly sought to intro-
duce this technique into the gene targeting technologies he had
already established.
WINTER 2 02 3 | T H E S C IE N T IST 1 5
 FOUNDATIONS

Across the world, Jamey Marth, a molecular and cellular biol-
ogist at the Biomedical Research Center, recognized the method’s
potential for modeling gene function. He wanted to study genes
that regulated protein glycosylation in animals for closer recapitu-
lation of the human system.
Marth recalled early discussions with his team members revolv-
ing around Sauer’s previous work with recombinases that cleaved
DNA in a very conservative and targeted manner in yeast and mam-
malian cells. Despite its success in mammalian cell lines, Marth
was worried that it would not translate well in an animal model. It
was possible that chromatin rearrangement during development
might shut down the prokaryotic activity of Cre. However, he was
undeterred and designed a Cre-expressing vector with two objec-
tives: obtain Cre expression, and make the mutation cell specific.
The results of his experiments surprised him.
He and his team demonstrated that Cre-loxP recombina-
tion efficiently deleted DNA sequences in specific developing
T cells of transgenic animals in 1992.10 “I remember the day we
saw the first autoradiograms coming off the machine,” recalled
Marth. “We were just overjoyed that this worked so well with
high efficiency.”
Around the same time, Marth received a call from Rajewsky.
Rajewsky wanted to collaborate after reading Marth’s paper on the
successful T cell cre transgenic line. Although Rajewsky initially
wanted to target the pol` gene in B cells, Marth’s T cell transgenic
line was an attractive alternative.

Cre-loxP recombination allows scientists to excise, insert, or invert specific DNA segments with unprecedented accuracy.
This works through two key components: a Cre recombinase and loxP sequence recognition sites. The Cre enzyme
identifies pairs of loxP sites (arrowheads), which flank (flox) the DNA, and catalyzes reciprocal DNA recombination
between the two sites to excise a small piece of DNA.

Q
A Q
B

To generate a tissue specific knockout mouse, researchers

breed a mouse bearing a Cre transgene under a tissue- or cell-
type specific or inducible promoter Q
A with a homozygous
floxed mouse Q B.
Target gene Target gene
Target gene Target gene
Tissue-specific promoter Cre IoxP site

Q
C Q
B

The offspring are heterozygous for the floxed

target gene Q
C and breed with the homozygous
floxed mouse Q B.

Target gene Target gene

Target gene Target gene
Tissue-specific promoter Cre

Q
D

The resulting experimental mouse is At tissue of interest:

hemizygous for Cre and homozygous for
loxP Q
D . This is the necessary genotype Deletion of gene
required to conditionally knock out the Target gene
target gene in the specific tissue.
Target gene

Tissue-specific promoter Cre Tissue-specific promoter Cre

TROUBLESHOOTING

MICROSCOPY
EXPERIMENTS

SPONSORED BY CUSTOM PUBLISHING BY

 TROUBLES

EXPERI
Fluorescence microscopy has revolutionized cell and
developmental biology research by enabling scientists to
visualize cellular processes in real time and dissect molecular
events in depth. However, researchers can encounter several
problems when imaging their samples through fluorescence
microscopy, including bleed-through, photobleaching, high
background signal, phototoxicity, and channel misalignment.
Fortunately, there are ways to overcome these major challenges.

BLEED-THROUGH

PROBLEM

Bleed-through is the phenomenon where the

emission from one fluorophore is detected in a
different fluorophore’s channel (above middle
image).1 This could lead scientists to erroneously
conclude colocalization between the fluorophores.

SPONSORED BY
SHOOTING
MICROSCOPY
MENTS

SOLUTION

Researchers avoid bleed-through artifacts by choosing

fluorophores with vastly different excitation and
emission spectra.1 Additionally, they can employ filters
with narrower wavelength ranges or spectral detectors
to avoid this problem in most cases.

CUSTOM PUBLISHING BY
 EXPERIMEN
PHOTOBLEACHING
Fluorescence microscopy has revolutionized cell and
developmental biology research by enabling scientists to
l time and
chers can
mples thro
ugh, phot
, and chan
ercome th

PROBLEM SOLUTION

Photobleaching is the phenomenon where Researchers reduce photobleaching by minimizing

BLEED-THROUGH
a cell’s fluorescent signal irreversibly
decreases over time because of
accumulating damage to the fluorophores.2
This process results from exposure to high
intensity excitation light.
the light source’s intensity or the exposure time.2
If the scientist is imaging the cells live, they
could increase the interval between time points,
while mounting media with antifade reagents
could help them prevent photobleaching for
fixed cell experiments. Moreover, they could
also use fluorophores requiring longer excitation
wavelengths, such as those excited in the near-
infrared region, to limit photobleaching.3

HIGH BACKGROUND SIGNAL OBLEM

Bleed-through is the phenomenon where the
emission from one fluorophore is detected in a
channel (abov
cientists to er
etween the flu

CUSTOM

PROBLEM SOLUTION

High background signal obscures the
fluorophore’s real signal and reduces the
apparent image resolution. This signal
is caused by out-of-focus fluorophores
detected above or below the focal plane,
cell autofluorescence, or incorrect staining.4
Researchers decrease the detection of out-of-focus
fluorescence by employing confocal microscopy over
wide-field microscopy to image thicker samples.5 They can
also reduce autofluorescence by choosing fluorophores
that have different spectra than the autofluorescence,
changing the fixative, or switching to a less autofluorescent
medium when imaging the cells live.4 Moreover, scientists
optimize sample staining by altering stain concentrations
or changing blocking reagents to diminish the background
signal and increase the apparent resolution.
 PHOTOTOXICITY
PROBLEM
Phototoxicity is the phenomenon where repetitive
exposure to the light source during live-cell
imaging causes damage to the cell that can lead
to its death.3 This process leads to the production
of reactive oxygen species (ROS), which react
with the cell’s biomolecules and organelles to
produce the damage. Phototoxicity produces cell
morphology changes, including blebbing, apoptotic
body formation, and nuclear fragmentation.3,6 This
phenomenon often accompanies photobleaching.
CHANNEL MISALIGNMENT
PROBLEM
Channel misalignment is a phenomenon where
images acquired for each fluorescent channel do
not perfectly align with one another when overlaid
(above right image). Scientists encounter this
problem when the labeled organelles or biomarkers
shift between the acquisition of each channel during
live-cell imaging.
SOLUTION
Like photobleaching, reducing the light source’s intensity,
decreasing the exposure time, or increasing the interval
between images can mitigate the phototoxic effects of
fluorescence microscopy, while employing fluorophores
requiring longer excitation wavelengths also minimizes
light-induced damage.3 Additionally, fluorophore choice
has a direct effect on the amount of ROS produced in
the cell after illumination, indicating that changing the
fluorophore could reduce phototoxicity during live-cell
imaging.
SOLUTION
Researchers can avoid channel misalignment by
employing a beam splitter and dual cameras, which
each capture a channel simultaneously.7 They can
also acquire the channels concurrently by using a
single camera with an emission image splitter, which
projects each channel onto a different portion of the
camera’s sensors.
Seeing Is Solving

Mouse cerebellum captured with a UPLXAPO40X objective on a FLUOVIEW™ FV4000 laser scanning confocal microscope. Sample
courtesy of Dr. Katherine Given, Principal Investigator, Neurobiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

At Evident, we understand the value of a vision.

We focus on developing advanced life science innovations to meet your
laboratory and research needs. Our line of microscopy and imaging solutions
is built with quality, clarity, and value in mind.
Let’s work together to create an ideal environment for exploring possibilities.
For more information, visit EvidentScientific.com
EVIDENT
48 Woerd Avenue, Waltham, MA 02453, 800-446-5967
At Evident, we are guided by the scientific spirit—innovation and
exploration are at the heart of what we do. Committed to making
people’s lives healthier, safer and more fulfilling, we support our
customers with solutions that solve their challenges and advance
their work; whether it’s researching medical breakthroughs, inspecting
infrastructure, or exposing hidden toxins in consumer products.
Evident Life Science empowers scientists and researchers through
collaboration and cutting-edge life science solutions. Dedicated to
meeting the challenges and supporting the evolving needs of its
customers, Evident Life Science advances a comprehensive range
of microscopes for pathology, hematology, IVF, and other clinical
applications as well as for research and education.

For more information, visit EvidentScientific.com.

References

1. North AJ. Seeing is believing? A beginners’ guide to practical pitfalls in

image acquisition. J Cell Biol. 2006;172(1):9-18.
2. Ettinger A, Wittmann T. Chapter 5 - Fluorescence live cell imaging. In:
Methods in Cell Biology. Vol 123. Academic Press; 2014:77-94.
3. Icha J, et al. Phototoxicity in live fluorescence microscopy, and how to avoid
it. BioEssays. 2017;39(8).
4. Waters JC. Accuracy and precision in quantitative fluorescence microscopy.
J Cell Biol. 2009;185(7):1135-1148.
5. Elliott AD. Confocal microscopy: Principles and modern practices. Curr
Protoc Cytom. 2020;92(1):e68.
6. Laissue PP, et al. Assessing phototoxicity in live fluorescence imaging. Nat
Methods. 2017;14(7):657-661.
7. Valli J, et al. Seeing beyond the limit: A guide to choosing the right super-
resolution microscopy technique. J Biol Chem. 2021;297(1):100791.

S
 • Are you submitting a manuscript
or a grant?

Scientific
• Trying to create an eye-catching
figure or poster?

Services
• Looking for a slide deck that will
capture the attention of the entire
conference hall?

Our Scientific Services team can help you shape your

message and deliver it to the people who need to see it.

EDITORIAL GRAPHICS COMMUNICATIONS

Our PhD-trained scientific Our professional graphic Our team can polish your
writers will sharpen your designers will bring your message and present it
manuscript's language and look. figures and schematics to life. to your audience.

Figure 3 Hia
1 BD2 BD1 1098

Hsf
1 BD2 BD3 BD1 2414
BEFORE

Signal peptide
Figure 3. Domain arrangements of Hia and Hsf trimeric auto- Neck/IsNeck domain
transporters. The Hia passenger domain is characterized by a Trp ring domain
repetitive architecture consisting of multiple domain types. Hsf GANG domain
KG connector domain
consists of a similar but extended domain arrangement com-
Ylhead domain
pared to Hia. Domain arrangements were obtained from the TTT domain
daTAA server (http://toolkit.tuebingen.mpg.de/dataa). -barrel domain

Signal Peptide Neck/IsNeck domain

Trp ring domain GANG domain Hia
1 BD2 BD1 1098
KG connector domain Y1head domain
TTT domain B-barrel domain

Hsf
1 BD2 BD3 BD1 2414 AFTER

Learn more at WWW.THE-SCIENTIST.COM/PAGE/SCIENTIFIC-SERVICES

 By crossing Marth’s T cell specific cre transgene with Rajew-
sky’s conditional pol` allele, Marth and Rajewsky developed mice
that lacked DNA polymerase ` in T cells.11 “When we did this work,
this kind of technology opened the way to do lots of things beyond
conditional targeting,” said Rajewsky.
Expanding the scope of Cre-loxP recombination
The success of Rajewsky’s and Marth’s conditional gene targeting
led to an explosion of different Cre transgenic lines with expression
profiles in various tissues. Researchers developed additional levels
of control over gene expression as a natural extension of this found-
ing technology. Mainly, researchers developed temporal control to
circumvent previous challenges that arose from global gene dele-
tion or early developmental Cre recombinase activity. The incor-
poration of drug- or interferon-responsive promoters allowed
researchers to control expression of Cre recombinase. Researchers
used tetracycline, type I interferon, or tamoxifen to induce pro-
moter activation.12-14
Andrew McMahon, a developmental biologist from the Uni-
versity of Southern California, and his postdoctoral researcher
Paul Danielian, who is now a biomedical editor at General
MODIFIED FROM © SHUTTERSTOCK.COM, GASPAR GOMES COSTA; © ISTOCK.COM, SEAMARTINI; DESIGNED BY ERIN LEMIEUX
Dynamics Information Technology, refined Cre recombinase
control in vivo with mice using tamoxifen. “With a founding
technology like Cre-loxP, a lot of resources got built around
that,” recalled McMahon.
Danielian previously studied steroid hormone regulation, and
his ideas guided their subsequent experiments. The duo fused the
ligand binding domain of the estrogen receptor to Cre to generate
a conditional form of it knowing that it would be sequestered in
the cytoplasm in the absence of a ligand. Adding a ligand activated
this fusion protein and translocated it to the nucleus to activate Cre
activity and induce recombination. This was the first time anybody
had conditionally removed gene activity in the developing fetus of
the mammalian system.15 Since then, tamoxifen-inducible Cre-loxP
has been one of the most widely used inducible systems.
“It’s just an illustration of how powerful genetics is. The more
you can exquisitely control the process that you’re working with,
the more insight you’re going to get into the question that you’re
asking,” said McMahon.
Combining cre-ations
The Cre-loxP system remains the gold standard method for condi-
tional gene regulation in mice, but it can be costly and time consum-
ing. So, over the last few years, researchers wondered whether to com-
plement this method with another powerful gene editing technique.
They found their opportunity with the discovery of clustered regularly
interspaced short palindromic repeats (CRISPR) and CRISPR associ-
ated nuclease, Cas9, which together enable highly specific DNA altera-
tions at precise locations within the genome.16
The Cre-loxP-CRISPR combination sparked researchers’ interest
in investigating its potential applications. CRISPR could introduce
specific mutations or genetic variations and the Cre-loxP recombina-
tion system could precisely excise or integrate the genetic elements.
For Stefan Hans, a developmental geneticist at the Dresden
University of Technology, this two-step approach offered the best
of both technologies for his studies on neuron regeneration in
zebrafish.17 The speed of transgenic zebrafish generation and the
ability to precisely visualize the target cells were key advantages.
“While you can be pretty sure that the cells are mutant, you also
need to know where your cells are to understand how they behave.
So, this feature is an important aspect because the putative mutant
cells are labeled, and we can easily identify and take out these cells
for analysis,” said Hans. Although the combination of these tech-
niques was demonstrated in mammalian cells a few years prior,
Hans was the first to use it in zebrafish research in 2021.18
The Cre-loxP system left a lasting influence on conditional
gene editing, and modern advances have taken gene function and
development to new heights. “Techniques come and go with new
technology, It’s just like night and day. So, when you have a tech-
nique that’s lasted 30 years with no replacement technology, I
think that’s kind of remarkable,” said Marth. J
References
1. Sternberg N. Demonstration and Analysis of P1 Site-specific Recombination Using
h-P1 Hybrid Phages Constructed In Vitro. Cold Spring Harb Symp Quant Biol.
1979;43:1143-1146.
2. Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination: I.
Recombination between loxP sites. J Mol Biol. 1981;150(4):467-486.
3. Saur B. Functional expression of the cre-lox site-specific recombination system in
the yeast Saccharomyces cerevisiae. Mol Cell Bio. 1987;7(6):2087-2096.
4. Saur B, Henderson N. Site-specific DNA recombination in mammalian
cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A.
1988;85(14):5166-5170.
5. Evans MJ, Kaufman MH. Establishment in culture of pluripotent cells from mouse
embryos. Nature. 1981;292:154-156.
6. Doetschman T, et al. Targetted correction of a mutant HPRT gene in mouse
embryonic stem cells. Nature. 1987;330:576-578.
7. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse
embryo-derived stem cells. Cell. 1987;51:503-512.
8. Kitamura D, et al. A B cell-deficient mouse by targeted disruption of the membrane
exon of the immunoglobulin μ chain gene. Nature. 1991;350:423-426.
9. Gu H, et al. Independent control of immunoglobulin switch recombination at
individual switch regions evidenced through Cre-loxP-mediated gene targeting.
Cell. 1993;73(6):1155-1164.
10. Orban PC, et al. Tissue- and site-specific DNA recombination in transgenic mice.
Proc Natl Acad Sci. 1992;89(15):6861-6865.
11. Gu H, et al. Deletion of a DNA Polymerase ` Gene Segment in T Cells Using Cell
Type-Specific Gene Targeting. Science. 1994;265(5168):103-106.
12. St-Onge L, et al. Temporal Control of the Cre Recombinase in Transgenic Mice by a
Tetracycline Responsive Promoter. Nucleic Acids Research. 1996;24(19):3875-3877.
13. Kühn R, et al. Inducible gene targeting in mice. Science.1995;269(5229):1427-1429.
14. Metzger D, et al. Conditional site-specific recombination in mammalian cells
using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A,
1995;92(15):6991-6995.
15. Danielian PS, et al. Modification of gene activity in mouse embryos in utero by a
tamoxifen-inducible form of Cre recombinase. Cell. 1998;8(24):1323-1326.
16. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial immunity. Science. 2012;337(6096):816-821.
17. Hans S, et al. Cre-Controlled CRISPR mutagenesis provides fast and easy
conditional gene inactivation in zebrafish. Nat Commun. 2021;12:1125.
18. Yang F, et al. CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific
Genetic Manipulation Tool. Mol Ther Nucleic Acids. 2017;7:378-386.
WINTER 2 02 3 | T H E S C IE N T IST 1 7
Some species remove up to 90 percent of their genomes during
development, but why or how this happens is still a mystery.

BY APARNA NATHAN, PhD

 arie Delattre, a biologist at the École Normale Supérieure
M de Lyon, has studied the nematode Mesorhabditis belari for
nearly a decade now. The microscopic worm first caught her
attention for its unconventional approach to reproduction, where
only a small fraction of offspring keep their male parent’s DNA.1
But more recently, she was looking at Mesorhabditis embryos
under a microscope and noticed something else that was odd:
in some embryos, the DNA was shattered into small fragments.
As she looked more closely across all stages of embryo devel-
opment, she noticed a pattern. At the one-cell stage, the DNA
MODIFIED FROM © ISTOCK.COM, BYAKKAYA
looked normal. It stayed intact as the first cell divided into two
cells and then three cells. But when the embryo reached the five-
cell stage, the DNA fragments suddenly appeared.
They started in the nucleus, then moved into the sur-
rounding cytoplasm, and after a few more rounds of cell divi-
sion, disappeared entirely. Delattre was intrigued. During this
brief point in the worms’ development, part of their genomes
appeared to vanish.
What she observed is a process carried out by dozens of other
species. Termed programmed DNA elimination, this process
allows organisms to delete specific portions of their genomes dur-
ing development. “It was a really random discovery,” said Delat-
tre. “Serendipity, I would say.” Her team’s deeper investigation
into this phenomenon, recently published in the journal Current
Biology, added Mesorhabditis nematodes to the list of species that
can carry out this mysterious process.2,3
Even after more than a century of research, there are still many
unanswered questions about programmed DNA elimination.
“You see that this thing happens very commonly, suggesting it
has an important biological role,” said Jianbin Wang, a biologist
at the University of Tennessee, Knoxville. “It’s just that we don’t
really know what that role is yet.”
Researchers around the world are working to answer these
questions. Many of them stumbled upon programmed DNA elim-
ination by accident, like Delattre, but now they’re hooked; their
research could offer a new understanding of the ever-changing
nature of the genome.
New technology takes on an old theory
An organism’s DNA starts off in one lonely cell containing the
germline DNA passed down from the previous generation.
Through many rounds of cell division, new somatic cells emerge
T
 that contain essentially identical copies of DNA and become the
building blocks of the organism.
In the 1880s at the Zoological Institute, cell biologist The-
odor Boveri studied a species of parasitic worms called Paras-
caris, which has a relatively large genome compared to other
worms— so large that its DNA was visible even through a primi-
tive 19th century microscope. He observed that a large chunk of
the germline genome was removed as somatic cells developed.3
More than 100 years later, more sophisticated molecular biol-
ogy assays revealed that this worm removed an astounding 89
percent of its 2.5-billion-base genome.
As those were still the early days of cell biology, Boveri
assumed that this was a normal part of development. But as sci-
entists looked for this process in more organisms, they realized
that programmed DNA elimination was not universal.
Early work focused on microscopic species, including var-
ious species of parasitic worms and single-celled organisms
called ciliates. Scientists learned much of what they know about
programmed DNA elimination by studying a family of para-
sitic worms called Ascaris, which removes around one-fifth of
its germline genome.3 In the 1980s, researchers finally found a
family of vertebrates, the hagfish, that removes between one-

Many species undergo programmed DNA elimination, a process where specific parts of the genome found
in the original sperm and egg cells are removed from the cells of the developing body. Different species use
varied cellular mechanisms to remove specific parts of their genomes. This process has recently been docu-
mented in worms in the Mesorhabditis genus, which eliminate approximately thirty percent of their DNA.

One-cell stage Cells divide

Kinetochore Metaphase
plate

Early in the process of Mesorhabditis development,
cells still carry the germline genomes from the gam-
etes that produced the first cell. As early as the two
or four-cell stage, the DNA begins to fragment.
Researchers can see this under a microscope, and
it’s one of the first signs that a cell might be prepar-
ing for programmed DNA elimination.
As the cells prepare to divide, the DNA assembles
into chromosomes. Normally, microtubules latch
onto these chromosomes via each chromosome’s
kinetochore proteins. However, some DNA frag-
ments lack kinetochores, so the microtubules have
nowhere to bind.
The chromosomes arrange themselves in pairs along the middle
of the dividing cell in a region called the metaphase plate. Without
microtubules to guide them there, the unattached DNA fragments
do not migrate to the metaphase plate and instead linger in the sur-
rounding areas of the cell. They will likely be targeted for elimination.
 fifth and one-half of its germline genome.4 More recently, stud-
ies have shown that nearly all songbirds appear to eliminate
parts of their germline genomes.5
“We are exposed to organisms that have programmed DNA
elimination every single day,” said Alexander Suh, an evolution-
ary biologist at the Leibniz Institute for the Analysis of Biodi-
versity Change and Uppsala University.
Recent technologies such as DNA sequencing have bol-
stered researchers’ efforts to probe this process. By com-
paring sequences of the genomes of germ cells and somatic
cells from the same organism, researchers can look for long
MODIFIED FROM © ISTOCK.COM, ROCCOMONTOYA DESIGNED BY ERIN LEMIEUX
In the last stage of cell division, the microtubules pull the pairs
of chromosomes apart, so that each new cell’s nucleus gets one
chromosome from each pair. The unattached DNA fragments
remain in the center of the dividing cell, where they will be ran-
domly pushed into one of the two new cells.
In a wild-type cell, you would never
see this. This would be a red flag.
—Marie Delattre, Ecole Normale Superieure de Lyon
stretches in the germline genome that are absent from the
somatic genome. These studies have shown that species can
eliminate anywhere between 0.5 percent and 90 percent of
their genomes.
Each new cell’s nucleus contains one full copy of After a few more cycles of cell division,
the somatic genome, while the other DNA frag- the DNA excluded from the nucleus likely
ments remain in the cytoplasm outside the nucleus. degrades in the cytoplasm, as seen in
Other species such as sea lampreys eliminate other worms.
whole chromosomes instead of DNA fragments.
During cell division, these chromosomes migrate to
the metaphase plate along with the other chromo-
somes, but do not migrate to the poles of the divid-
ing cell. This behavior is known as “lagging” and
causes the chromosomes to be excluded from the
new cells’ nuclei and eliminated.
 Scientists have used sequencing to investigate exactly which
parts of the genome are removed and what instructions they
encode. Typically, the same regions are removed in every cell of an
organism and in every member of a species, although there are dif-
ferences between species. However, regardless of species, the elimi-
nated regions include large stretches of repeated DNA sequences,
which typically do not encode the instructions for proteins.
How to ditch DNA
Programmed DNA elimination pops up on almost every
branch of the tree of life, but the processes are as diverse as
the flora and fauna that use them. The nematodes and uni-
cellular ciliates seem to slice their genomes into small pieces
and remove a subset. Vertebrates seem to be more likely to
remove full chromosomes.
Understanding the origins of DNA
elimination is going to tell us a lot
piece of DNA at the tip of the chromosome appeared to drag the
chromosome in the opposite direction of the microtubule. As a
result, these lagging chromosomes get left behind when the nuclei
form and end up degraded in small pockets of the cytoplasm.7
Researchers still don’t know how the lagging chromosomes
or discarded DNA fragments are chosen. In worms, Delattre uses
detailed maps of the genome and RNA measurements to figure
out how the cell knows where to fragment the DNA and what pro-
teins make the cuts. Once she finds a compelling protein candi-
date, she hopes to delete it from an embryo and observe whether
the cells still undergo programmed DNA elimination.
Extreme DNA silencing
There’s another major open question: why would species want
to eliminate their DNA at all? Removing DNA is an extreme
way of keeping genes from being used in cells, according to
Jeramiah Smith, a biologist at the University of Kentucky. But
cells have other ways of doing this that are less disruptive. For
example, they can pack their DNA so tightly that the genes can’t
be accessed, or they can use small pieces of RNA that bind to

about how our own genome works.

—Jeramiah Smith, University of Kentucky

These different approaches involve the same set of core steps:

part of the genome is marked for elimination, and as the cell
divides, this DNA is shunted out of the nucleus and ultimately
removed from the cell. In ciliates and worms, the cell also needs
to slice up the DNA into fragments. How this happens in cells is
still largely unknown. But studies from Wang, Delattre, and oth-
ers are starting to piece together the process.
Cell division is a carefully orchestrated dance, where duplicated
chromosomes pair up in a double-file line in the metaphase plate
at the center of the cell. Long microtubules anchored at opposing
ends of the cell latch onto the kinetochore protein at the center of
each chromosome. As the cell divides, the microtubules reel in half
of the chromosomes to each new cell. In Ascaris, Wang showed
that DNA fragments that are ultimately eliminated actually lack
kinetochore proteins, so the microtubules can’t bind to them and
pull them into the new cells’ nuclei.6
Delattre saw a similar dearth of kinetochore proteins on elimi-
nated DNA in Mesorhabditis.2 Using a fluorescent label, she visual-
ized DNA fragments as tiny dots floating in a ring around the rest of
the DNA. The untethered DNA pieces get pushed to the perimeter
while the remaining chromosomes segregate. As the cell continues
dividing, the fluorescent dots end up outside the nucleus in the cyto-
SIMON BIANCHETTI

plasm, and ultimately fade away.

In vertebrate species, where whole chromosomes are elimi-
nated, things go awry when microtubules start pulling the chro-
mosomes apart. Certain chromosomes move slower than the oth- By looking at Mesorhabditis belaris embryos under a microscope, Marie
ers, a phenomeno≠n called lagging. In a study of sea lampreys, a Delattre noticed that they removed portions of their genomes.

22 T H E SC I EN T I ST | the-scientist.com

genes and inhibit protein expression. Species that carry out
programmed DNA elimination typically have the machinery to
silence genes in these ways too.
Another explanation is that eliminated genes might play a
role in germ cells during reproduction, where they may need to
make an arsenal of proteins that are unnecessary in other cells
of the body. Studies in Ascaris and in zebra finches revealed that
their eliminated genes have functions in sex organs like the tes-
tes, where germ cells originate.8,9
Not all researchers are convinced. In Mesorhabditis, Delat-
tre showed that the eliminated genes seem to follow no pat-
tern and aren’t critical to species survival.2 She thinks that in
these worms, the process could mainly serve to remove repeated
sequences, and any genes that are removed are just bystanders.
It’s also possible that programmed DNA elimination could
play diverse r oles in different species. “I would say, at this point,
that they’re different processes,” Suh said. Smith agreed and
noted that the evolutionary history of DNA elimination is still
hazy. He speculated that each major branch of life may have
independently developed the ability to eliminate DNA. “They’re
MARIE DELATTRE
doing similar things, but they got there through very different
evolutionary trajectories,” he said.
Although programmed DNA elimination hasn’t been
observed in humans, similar processes can occur in human
Scientists observe Mesorhabditis belari worms at various stages of devel-
opment through a microscope to study programmed DNA elimination.
cells, but typically only when they are malfunctioning. “In a
wild-type cell, you would never see this,” Delattre said. “This
would be a red flag.”
When human DNA breaks into fragments, a process called
chromothripsis, it’s a cellular event so catastrophic that it can
cause cancer. When human chromosomes don’t divide into
cells properly during embryonic development, we end up with
developmental disorders. “Understanding the origins of DNA
elimination is going to tell us a lot about how our own genome
works,” Smith said.
For example, it might help us better understand how these
events occur in cancer cells. Knowing how DNA is marked for
deletion and being able to do that artificially could even be
used as a therapeutic option for disorders caused by extra cop-
ies of chromosomes, such as Down syndrome.
For now, many scientists agree that the next step is to study
programmed DNA elimination in more species. “We probably
don’t know the majority of species that actually do this,” Smith said.
Suh and his team are doing just that. So far, they have
sequenced germline and somatic DNA from around 30 spe-
cies of songbirds. Every songbird eliminates at least one chro-
mosome, but in some cases it’s the biggest chromosome; in
other species it’s the smallest one. The genes on the eliminated
chromosomes can be completely different between species but
they have one thing in common: Some of the gene sequences
are very similar to those found on retained chromosomes, a
trend that hasn’t yet been observed in other types of animals.9
“It gets more and more confusing with every species,” Suh said. J
References
1. Grosmaire M, et al. Males as somatic investment in a parthenogenetic
nematode. Science. 2019;363(6432):1210-1213.
2. Rey C, et al. Programmed DNA elimination in Mesorhabditis nematodes. Curr
Biol. 2023;33(17):3711-3721.
3. Zagoskin MV, Wang J. Programmed DNA elimination: silencing genes and
repetitive sequences in somatic cells. Biochem Soc Trans. 2021;49(5):1891-
1903.
4. Smith JJ, et al. Programmed DNA Elimination in Vertebrates. Annu Rev Anim
Biosci. 2021;9:173-201.
5. Torgasheva AA, et al. Germline-restricted chromosome (GRC) is widespread
among songbirds. Proc Natl Acad Sci USA 2019;116(24):11845–11850.
6. Kang Y, et al. Differential Chromosomal Localization of Centromeric Histone
CENP-A Contributes to Nematode Programmed DNA Elimination. Cell Rep.
2016;16(9):2308-2316.
7. Timoshevskiy VA, et al. Germline-Specific Repetitive Elements in
Programmatically Eliminated Chromosomes of the Sea Lamprey (Petromyzon
marinus). Genes (Basel) 2019;10(10):832.
8. Wang J, et al. Silencing of Germline-Expressed Genes by DNA Elimination in
Somatic Cells. Dev Cell. 2012;23(5):1072-80.
9. Kinsella CM, et al. Programmed DNA elimination of germline development
genes in songbirds. Nat Commun. 2019;10(1):5468.
WINTER 2023 | T H E S C IE N T IST 2 3
 The h al
L e of e lacenta
in g e np
o p l s ep p
Y E , D
W e have all had one, and we owe our lives to it. It’s the
first organ to develop and it simultaneously serves
as the lungs, kidneys, immune system, and digestive
tract, to name a few, in a fetus while it develops these systems.
Despite being one of the most important organs, the placenta is
one of the least understood.
“It’s such a fascinating organ,” said Norah Fogarty, a devel-
opmental biologist at King’s College London. “We know so lit-
tle about it, but there’s also this kind of intrigue about the pla-
centa.” This mysterious organ has inspired lore and customs
for centuries.
Throughout gestation, the fetus depends entirely on the pla-
centa. The discoid-shaped organ serves as a barrier between the
parent and child. Although some researchers describe the pla-
centa as an evolutionary battleground due to the mix of mater-
© SHUTTERSTOCK.COM, SCIEPRO
nal and paternal DNA both vying for resources, it is also a space
where compromise prevails to ensure the health of both par-
ties. Research has linked abnormal placental development to a
number of pregnancy complications, including preeclampsia,
fetal growth restriction, placental abruption, and preterm labor,
collectively referred to as the great obstetric syndromes.1 Pre-
eclampsia, characterized by high blood pressure and increased
protein in the urine after 20 weeks of pregnancy, occurs in 3-5%
tood organ,
of pregnancies.2 Preeclampsia ranges from mild to severe and
can be life-threatening for the parent and child. Currently, the
only cure is delivery of the baby and placenta.
The dearth of treatments for preeclampsia stems from a
greater gap in our knowledge. “We also don’t know what’s hap-
pening in normal placenta development,” said Fogarty. The lack
of physiologically relevant models of placental development
stymies efforts to close these knowledge gaps. Although animal
models have provided valuable insight into the organ’s devel-
opment, the placenta is one of the most evolutionarily diver-
gent organs, and considerable differences in the developmen-
tal trajectory, morphology, and degree of placental invasion
into the uterine wall demand caution when extrapolating data
from other species to humans.3 Additionally, several ethical and
logistical obstacles hinder the study of early placenta develop-
ment in humans.
These obstacles led researchers to develop in vitro models, but
until recently it wasn’t clear how robust these models could be.
“The placenta has been difficult to capture in the dish,” said Fog-
arty. Now, a few key advancements in cell culture techniques over
the last decade have breathed new life into the field, and many
hope that these models hold the key to unlocking the secrets of
the space between.
WINTER 2 02 3 | T H E S C IE N T IST 2 5
 A black box
Following fertilization, one cell becomes two, and those become
four and so on, until the zygote transforms into a blastocyst
around six days post fertilization (dpf ).4 The blastocyst, or the
preimplantation embryo, comprises of an inner cell mass (ICM)
swaddled by an outer layer of cells that make up the trophecto-
derm. The trophectoderm, which is home to nearly 90 percent
of the blastocyst cells, develops into the placenta while the ICM
gives rise to the fetus.
Fogarty wants to understand the molecular processes that
orchestrate these early developmental stages. As an undergradu-
ate student at Trinity College Dublin, Fogarty’s interest in fetal
health and development began when she enrolled in a course on
molecular medicine that focused on treating diseases of adult-
hood. “It led me to think ‘you know, we’re focusing all this time
and research into treating diseases in the adult, but if we can help
babies be born as healthy as possible and grow up to be healthy
adults, then we would likely eradicate a lot of these diseases,’”
said Fogarty.
Near the end of her studies, she came across an email that
piqued her interest: It was an advertisement about a PhD project on
placenta development at the University of Cambridge. She applied
and got the position where she studied transcriptional dynamics in
the human placenta under the joint supervision of Graham Burton
and Anne Ferguson-Smith. Following her doctoral studies, Fog-
arty joined the lab of stem cell and developmental biologist Kathy
Niakan at the Francis Crick Institute to continue her investigations
into the molecular drivers of early cell fate.
Transcription factors orchestrate trophectoderm develop-
ment and differentiation. Using comparative analyses, research-
ers previously demonstrated that two such factors, octamer-bind-
ing transcription factor 4 (OCT4) and caudal-type homeobox-2
(CDX2), exhibit temporally and spatially distinct expression
patterns in the embryos of mice and humans.5 Considering the
divergent expression patterns between the two species, Fogarty
was curious about the function of OCT4 during human embryo
development. To study this, she turned to CRISPR-Cas9-medi-
ated genome editing. Deletion of the gene encoding OCT4 from
early zygotes donated from patients of IVF clinics led to a down-
regulation of trophectoderm genes, including CDX2, and compro-
mised the development of the blastocyst.6 In contrast, when the
researchers manipulated mouse embryos in a similar manner, the
blastocyst formed but its maintenance was compromised. Fogar-
ty’s research detailed functional consequences of species-specific
gene expression patterns, further illustrating why mouse models
may fail to capture key developmental events in humans.
Around six to seven dpf, the blastocyst implants into the sur-
face of the uterine wall and begins its expansion.4 This area of the
endometrium is transformed early on in pregnancy and acts as a
fluffy bed in which the embryo grows. As the blastocyst burrows,
the trophectoderm begins to differentiate into subtypes of tro-
phoblast cells, starting with cytotrophoblasts, which are progeni-
tor stem cells in the placenta that give rise to other trophoblasts.
26 T H E SC I EN T I ST | the-scientist.com
This point in development, approximately 14 dpf, corresponds
to around the time of the first missed period, and it is typically
the earliest point that most people realize that they are pregnant.
Early zygote and blastocyst donations from patients of IVF clinics
have helped shed light on this black box period of development,
but human embryos are a limited resource and ethical concerns
restrict their long-term use in the lab. These earliest days of devel-
opment, although temporally distant from the clinical manifesta-
tion of preeclampsia, may lay the foundations for future problems.
“In the last few years, there have been more hypotheses being
developed that it’s a defect in the cytotrophoblast cell that sets the
track for whether preeclampsia will develop or not,” said Fogarty.
“We also don't know what's
happening in normal placenta
development.”
—Norah Fogarty, King’s College London
There are a number of available in vitro models for the study
of human placental development with varying degrees of physi-
ological relevance.2,4,7 Choriocarcinoma cells, derived from malig-
nant tumors of trophoblasts, are genetically abnormal and mouse
trophoblast stem cells, while a valuable tool, do not fully recapitu-
late the genetic and molecular milieu orchestrating human pla-
cental development.
However, 2018 saw a major breakthrough: For the first time,
researchers successfully generated bona fide human trophoblast
stem cells (hTSC).8 The researchers demonstrated that either troph-
ectoderm from the blastocyst or first-trimester placentas could be
used to generate bipotent trophoblast stem cells. Now, researchers
finally have access to pure trophoblast cells with the capacity for
self-renewal. By tweaking what they feed the hTSC, researchers can
transform the cells into different trophoblast subtypes.
The hTSC are useful models for studying trophoblasts, while
the embryo provides unrivaled access to studying trophectoderm
development. Fogarty uses the hTSC alongside human embryos
to study the signaling pathways that regulate trophectoderm and
early trophoblast development and differentiation. Furthermore,
hTSC are a useful platform for optimizing tools and develop-
ing hypotheses before testing them in valuable human embryos.
“These experiments will further our understanding of hTSC and
how they differ from the trophectoderm, but will also give us
insights into trophoblast biology,” said Fogarty. She hopes that
these tools can one day help reveal how defects emerging early in
development set the stage for placental diseases like preeclampsia.
Miniplacentas in a dish
As the invasion into the uterine wall wages on, cytotrophoblasts dif-
ferentiate into syncytiotrophoblasts (SCT), which carve out villi, or
 frond-like structures that soon house the fetal capillary system.7 As
SCT build larger and larger villi, cytotrophoblasts march forward
to conquer a new frontier in search of nutrients to fuel the contin-
ued expansion. These rogue cytotrophoblasts go deeper into the
uterine wall and differentiate into incredibly invasive extravillous
trophoblasts (EVT). Once there, EVT hunt down uterine arteries,
enlarge them, and hook them up to the placenta. Finally, around
10 weeks into the pregnancy, the parental circulation reaches the
intervillous space.9 By 12 weeks, the placental blueprint is in place.
The uterine wall is home to glands, vessels, stromal cells, and
immune cells that interact with the invading fetal cells to cre-
ate a boundary between the parent and fetus.4 The relationship
between the parent and the growing fetus is often portrayed as
parasitic or antagonistic, a 9-month war waged from within. This
is due in part to the highly invasive nature of EVT leaching nutri-
ents, but also the presence of the fetus’ foreign DNA. But Ashley
Moffett, a reproductive immunologist at the University of Cam-
bridge, said that the relationship between the parent and the pla-
centa isn’t simply friend or foe. “It’s a compromise, actually.”
Moffett, a doctor and pathologist by training, didn’t set out
to study the placenta. In the 1980s, she was looking for a job at
the hospital in Cambridge. The only available job at the time was
in the maternity ward. “I was sort of banished to the maternity
hospital without any interest at all in this, but I then, of course,
realized that there were these major disorders and that they were
completely understudied,” recalled Moffett. “Nobody knew any-
thing about them really.”
PAULA BALESTRINI
While working in the maternity ward, Moffett recalled influ-
ential papers published in the early 1980s that suggested that pre-
eclampsia results from a failure of EVT to properly invade the uter-
ine wall.1 She also remembered some peculiar looking cells that she
came across in pathology training. “I looked at every single organ in
the body under the microscope,” said Moffett. “I realized that there
were some cells in the uterus that I’ve never seen anywhere else.”
She thought that they might be a kind of natural killer (NK)
cell, so Moffett contacted Charlie Loke, one of her undergradu-
ate professors from the University of Cambridge and an expert
in reproductive immunology, to study these cells in the lab. After
only a month in the lab they figured out that these cells were, in
fact, a type of NK cell. “And [Loke] said, ‘you know, you’ll never
go back to clinical medicine,’ and I didn’t ever go back to clinical
medicine,” said Moffett. “That was the end of my medical career.”
Uterine NK cells, which differ substantially from blood NK
cells, dominate the immune cell landscape of the uterine wall bor-
dering first trimester placentas.10 Moffett and others went on to
characterize this unique immune cell and demonstrate its impor-
tance as a mediator between the needs of the mother to retain
resources and the needs of the baby to grow.
To explore the boundary between the parent and fetus, Moffett
and her team used single-cell RNA sequencing on placental and
endometrial samples donated by patients who underwent elec-
tive pregnancy termination in the first trimester.11 They identified
transcription factors that orchestrate cytotrophoblast differenti-
ation into SCT or EVT but also uncovered three subtypes of NK
cells with distinct immune regulation and cell-cell communica-
tion profiles. Their findings further highlighted the compromise
between parent and fetus, suggesting that NK cells keep a check
on EVT expansion while these cells protect the fetus from paren-
tal immune responses.
Around the same time in 2018, Moffett and her team and Mar-
tin Knofler’s research team at the Medical University of Vienna
separately published the first organoid models for trophoblasts.12,13
These three-dimensional organoid models offered another step
towards a physiologically relevant model that recapitulates cer-
tain aspects of the in vivo environment. To build a mini-placenta,
the researchers isolated proliferative cells from first trimester pla-
centa tissue and cultured them in a special cocktail chock full of
growth factors that coax trophoblast development and assembly
into a three dimensional blob of cells. Not only did the tropho-
blast organoids retain transcriptomic and methylation patterns
characteristic of in vivo first trimester trophoblasts, but they also
developed hormone-secreting SCT with intricate structures akin
to villi as well as migratory EVT. These self-replicating mini-pla-
centas even produced enough of the hormone chorionic gonado-
tropin to test positive on an at-home pregnancy test.12
In her lab at King’s College London, Norah Fogarty studies transcriptional
events that orchestrate early placenta development.
WINTER 2 02 3 | T H E S C IE N T IST 27
EARLY PLACENTA
DEVELOPMENT SETS
THE STAGE
During early pregnancy, the placenta remodels the uterine environment to support fetal growth.

DAY 0
DAYS 10-12
By 12 dpf, cytotrophoblast cells begin
to penetrate the primitive syncytium
to form primary villi, which later
DAY 1
form the villous placenta.

Primary villi
DAY 2
Primitive
syncytium

DAYS 7-9
After implantation, the trophectoderm
starts reshaping the endometrium.
A layer of cytotrophoblasts—tropho-
DAY 3
blast progenitor cells—emerges
around the same time as the invading
primitive syncytium.

DAY 4
DAYS 6-7 Cytotrophoblasts

Six to seven dpf, the blasto-

Inner cell mass
cyst attaches to the uterine
wall and begins its invasion.

Blastocyst

Trophectoderm
DAYS 5-6
Approximately five days post fertilization
(dpf), the blastocyst develops. The inner
cell mass gives rise to the fetus, while the
surrounding trophectoderm transforms
into the placenta.

-scientist.com
 From weeks three to 10, cytotrophoblast cells escape into the decidua,
Decidua
a specialized layer of endometrium, and differentiate into extravillous
trophoblasts. These invading cells remodel spiral arteries to reroute
parental blood to the intervillous space.

EARLY FIRST TRIMESTER PLACENTA

Villus

Syncytiotrophoblast

Cytotrophoblast

By the beginning of the second trimester, the cyto-

trophoblast plug breaks down and parental blood Spiral artery
begins to enter the intervillous space.
Extravillous
trophoblast
© JULIA MOORE, WWW.MOOREILLUSTRATIONS.COM

FULL TERM PLACENTA

BEYOND WEEK 10
PLACENTA

WINTER 2023 | T H E S C IE N T IST 2299

 In the 1980s, Moffett gained access to rare first trimester preg-
nancy hysterectomies, which included the entire uterus. She safely
tucked these samples away with the hope that one day new tools
would emerge to explore their cellular intricacies. Earlier this year,
Moffett returned to her historical hysterectomy samples and pub-
lished a spatially resolved multiomics single-cell atlas that captures
the trajectory of trophoblast differentiation as the cells invade and
transform the arteries in the uterine wall.14 This rich resource iden-
tified transcription factors and key cell-cell interactions, including
uterine NK cells in close proximity with EVT. Furthermore, they
found many of the same factors expressed on EVT derived from
hTSC and primary trophoblast organoids. “We now have a trajectory
of the whole invading trophoblast in humans for the first time, and
I think the organoids do recapitulate that quite well,” said Moffett.
After a long career, Moffett recently handed over the keys to
her lab, but she hopes these organoids will go on to provide a
relevant platform for studying important placental biology ques-
tions that have relevance to placental disorders like preeclampsia.
I realized that there were some
cells in the uterus that I’ve never
seen anywhere else.
—Ashley Moffett, University of Cambridge
Shallow roots
As the pregnancy progresses, cytotrophoblast cells keep dividing,
and the placenta keeps getting bigger and bigger to keep up with
the needs of the growing fetus. Shallow implantation of EVT early
in development might be one cause of preeclampsia.1 This results
in an insufficient or poor transformation of the arteries and paves
the way for a sparsely branched villous tree and weak perfusion
network for blood and waste products to travel between the par-
ent and fetus.7 This can cause serious problems later in pregnancy.
Mariko Horii trained as an obstetrician in Japan before com-
ing to the University of California, San Diego in 2013 to work with
Mana Parast, a placental pathologist. Horii arrived searching for
answers to why the placenta grows so poorly as has devastating
consequences for some of her patients. At the time, Parast was
gearing up to develop in vitro models for studying preeclampsia.
A lot of what researchers in the field know about human pla-
cental development comes from morphological, immunohisto-
chemical, and transcriptomic analyses of primary first-trimester
placental tissue.4 While an incredibly rich source of information,
access to these tissues is limited, and isolated cells did not survive
for long in a dish, making it difficult to run experiments in the lab.
This left scientists with a choice between genetically abnormal
cancer cell lines or mouse trophoblast stem cells for their research.
A major advance came in 1998 when James Thomson, a stem cell
biologist at the University of Wisconsin-Madison, successfully iso-
30 T H E SC I EN T I ST | the-scientist.com
lated stem cells from human blastocysts.15 Shortly after, his research
team demonstrated that they could differentiate human embry-
onic stem cells (hESC) into hormone-secreting cells akin to SCT by
feeding the cells a special media spiked with bone morphogenetic
protein-4 (BMP4).16 The subsequent development of induced plu-
ripotent stem cells (iPSC) via the reprogramming of somatic cells
provided scientists with a less controversial, more accessible source
of human pluripotent stem cells.17 Since then, Horii and others have
demonstrated that both hESC and iPSC can transform into tropho-
blasts and subtypes of trophoblasts by altering the environmental
conditions and feeding the cells different molecular cocktails.7,18
While the advent of hTSC and trophoblast organoids in 2018
are major stepping stones, they come with limitations. “Since we
have primary cells, then why not use the primary cells for a model
system instead?” said Horii. Scientists are still struggling to produce
hTSC and trophoblast organoids from full term placentas and cur-
rently derive them from either blastocysts or first trimester placen-
tas. Both sources are limited and, in some countries, laws restrict
their use. Horii raised another limitation of using cells sourced from
early pregnancy. “We don’t have the scientific knowledge to pre-
dict from the early first trimester pregnancy materials whether the
patient would have developed pregnancy complications or not.”
To build this knowledge, Horii and her team turned to full term
placentas for iPSC. Either mesenchymal stem cells derived from the
umbilical cord or cytotrophoblasts can transform into iPSC when
fed a special cocktail.17 Using iPSC derived from placental cells,
Horii and others have worked doggedly to refine culture protocols
over the years to generate trophoblasts.7,18 Eventually, research-
ers working with these cells demonstrated that their putative tro-
phoblasts secreted key hormones and expressed the EVT marker
HLA-G alongside other key genes expressed by trophoblasts.19,20
Recently, Horii and her team modified their protocol to include
a WNT-inhibitor alongside BMP4 to ensure the exclusion of meso-
derm cells and differentiation in trophoblast cells resembling cyto-
trophoblasts.21 However, they struggled to maintain primed stem
cells, or iPSC-derived trophoblasts, in a state of self-renewal. To fix
this, Horii and her team fed their iPSC the usual fare of BMP4 plus
a WNT-inhibitor but swapped the main culture media for one they
whipped up for the newfangled hTSC.22 “We were able to finally
derive the self-renewing trophoblast stem cells,” said Horii. They
further differentiated their new and improved cytotrophoblasts into
EVT or SCT using cell type specific differentiation protocols.
Horii thinks that the iPSC-derived trophoblast models will be
particularly useful for disease modeling because scientists can use
iPSC to produce cell types beyond trophoblasts, like blood vessels
or stromal cells. Currently, in her own lab, she uses this revamped
protocol on cells isolated from term placentas of patients with pre-
eclampsia. Her prior work using an earlier version of the culture
protocol suggested that this will be a fruitful avenue for modeling
preeclampsia in a dish. iPSC derived from placentas of pregnancies
with preeclampsia recapitulate several defects observed in primary
placenta tissues, including failure to respond to changes in sur-
rounding oxygen levels and abnormalities in EVT differentiation.23

Six days after fertilization, the human embryo holds epiblast cells (red) and
the trophectoderm (green). Epiblast cells go on to form the fetus, while the
trophectoderm gives rise to the placenta.
Fleeting but indelible
Following the birth of the baby comes the birth of the placenta as
it sheds away from the lining of the uterus. By the end of gesta-
tion, the SCT region is incredibly invaginated and convoluted to
provide a large surface area for diffusion to the baby. “If you were
to spread it out it would be 13 square meters in size,” said Fogarty.
That’s about the size of a parking space.
Just like that, this transient organ that helped the fetus sur-
vive in the womb for the last nine months is gone. Scientists are
increasingly appreciating the link between the in utero environ-
ment, including placental health, and susceptibility to chronic dis-
eases later in life.24 For example, babies that are born too big or too
small relative to their growth potential are at a higher risk for devel-
oping cardiovascular disease, diabetes, and obesity in adulthood.
These long-lasting effects further emphasize the need for improved
screening, prevention, treatment options, and of course, physiologi-
cally robust and relevant models.
“We now have the tools,” said Fogarty. Trophoblast stem cells,
trophoblast organoids, iPSC-derived trophoblasts, extended
embryo culture, CRISPR Cas9-mediated genome editing,
advanced imaging technology, scRNAseq, and spatial transcrip-
tomics all have a role to play in the study of the placenta.
While no model perfectly captures the complexities of this
mysterious organ, recent advances, including refined culture pro-
tocols and new in vitro systems, will facilitate the continued study
of human placentation. Some researchers are even developing pla-
centa-on-a-chip models using human iPSC-derived trophoblasts
to study placental perfusion dynamics.25
“There’s a renewed interest in the field, an energy in the field, that
will allow us in the next 10-20 years to make these breakthroughs
NORAH FOGARTY
and bring our understanding of the placenta up to speed with a lot of
the other organs that we know so much about,” said Fogarty.
“Just over half of all pregnancies are uncomplicated, normal
pregnancies,” said Fogarty. The other half are affected by miscar-
riage, intrauterine growth restriction, fetal growth restriction, and
preeclampsia. “If we can make these insights, there’s going to be
massive numbers of patients who can potentially be helped in the
future. There’s potential to make a big impact.” J
References
1. Brosens I, et al. The “great obstetrical syndromes” are associated with disorders
of deep placentation. Am J Obstet Gynecol. 2011;204(3):193-201.
2. James JL, et al. Modelling human placental villous development: Designing
cultures that reflect anatomy. Cell Mol Life Sci. 2022;79(7):384.
3. Roberts RM, et al. The evolution of the placenta. Reproduction.
2016;152(5):R179-189.
4. Turco MY, Moffett A. Development of the human placenta. Development.
2019;146(22):dev163428.
5. Niakan KK, Eggan K. Analysis of human embryos from zygote to blastocyst
reveals distinct gene expression patterns relative to the mouse. Dev Biol.
375(1):54-64.
6. Fogarty NME, et al. Genome editing reveals a role for OCT4 in human
embryogenesis. Nature. 2017;550:67-73.
7. Horii M, et al. Modeling human trophoblast, the placental epithelium at the
maternal fetal interface. Reproduction. 2020;160(1):R1-R11.
8. Okae H, et al. Derivation of human trophoblast stem cells. Cell Stem Cell.
2018;22(2):50-63.e6.
9. Jauniaux E, et al. Onset of maternal arterial blood flow and placental oxidative
stress. Am J Pathol. 2000;157(6):2111-2122.
10. Moffett A, Colucci F. Uterine NK cells: Active regulators at the maternal-fetal
interface. J Clin Invest. 2014;124(5):1872-1879.
11. Vento-Tormo R, et al. Single-cell reconstruction of the early maternal-fetal
interface in humans. Nature. 2018;563:347-353.
12. Turco MY, et al. Trophoblast organoids as a model for maternal-fetal
interactions during human placentation. Nature. 2018;564:263-267.
13. Haider S, et al. Self-renewing trophoblast organoids recapitulate the
developmental program of the early human placenta. Stem Cell Reports.
2018;11(2):537-551.
14. Arutyunyan A, et al. Spatial multiomics map of trophoblast development in
early pregnancy. Nature. 2023;616:143-151.
15. Thomson JA, et al. Embryonic stem cell lines derived from human blastocysts.
Science .1998;282(5391):1145-1147.
16. Xu R-H, et al. BMP4 initiates human embryonic stem cell differentiation to
trophoblast. Nat Biotechnol. 2002;20:1261-1264.
17. Takahashi K, et al. Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell. 2007;131(5):861-872.
18. Roberts RM, et al. Specification of trophoblast from embryonic stem cells
exposed to BMP4. Biol Reprod. 2018;99(1):212-224.
19. Amita M, et al. Complete and unidirectional conversion of human
embryonic stem cells to trophoblast by BMP4. Proc Natl Acad Sci USA.
2013;110(13):E1212-1221.
20. Horii M, et al. Human pluripotent stem cells as a model of trophoblast
differentiation in both normal development and disease. Proc Natl Acad Sci
USA. 2016;113(27):E3882-E3891.
21. Horii M, et al. An improved two-step protocol for trophoblast differentiation of
human pluripotent stem cells. Curr Protoc Stem Cell Biol. 2019;50(1):e96.
22. Soncin F, et al. Derivation of functional trophoblast stem cells from primed
human pluripotent stem cells. Stem Cell Reports. 2022;17(6):P1303-1317.
23. Horii M, et al. Modeling preeclampsia using human induced pluripotent stem
cells. Sci Rep. 2021;11(1):5877.
24. Thornburg KL, Marshall N. The placenta is the center of the chronic disease
universe. Am J Obstet Gynecol. 2015;213(4):S14-S20.
25. Lermant A, et al. Development of human iPSC-derived placental barrier-on-
chip model. iScience. 2023;26(7):107240.
WINTER 2 02 3 | T H E S C IE N T IST 3 1
 Unraveling
the Mystery
of Zombie Genes
Digging into how and why some genes are resurrected after death sounds morbid,
but it has practical applications.

BY IRIS KULBATSKI, PhD

32 T H E SC I EN T I ST | the-scientist.com
MODIFIED FROM © ISTOCK.COM, ILYA LUKICHEV

LI
ife begins and ends in low oxygen. Mammalian
embryos are submerged in a hypoxic environment
before the cardiovascular system and placenta
develop. In this low oxygen state, embryonic stem
cells hum with activity. They proliferate, activate
developmental genes, and transcribe DNA in an intri-
cate dance that choreographs the first buds of existence.1,2 When
this early mass of pluripotent stem cells, the blastocyst, bur-
rows into the lining of the uterus, access to higher oxygen levels
through the maternal blood supply triggers stem cells to differ-
entiate into cells that form the various tissues and organs.3,4 The
genes that initiate and wind the clock of life eventually go dor-
mant as key developmental milestones are reached.
The will to live
That’s life. But what about death? Less than a decade ago,
researchers debunked the long-held assumption that gene
expression—a hallmark of life—ceases at the time of death.
While most gene activity is extinguished after an organism dies,
certain zombie genes are reawakened, sometimes days later.
Some of these are the very same genes that are active during
development, then repressed throughout an organism’s lifetime.
Death also activates other genes involved in mechanisms such
as cell stress responses,, inflammation, immunity, and cancer.5,6
Why and how their resurrection occurs remains a mystery.
Cell death is a natural and essential part of the biological life
cycle. During the dance of development, cell death choreographs tis-
sue maturation and corrects developmental errors.7 Cell death also
plays an important role in the body’s response to cancer by mitigating
genetic mutations and uncontrolled cell proliferation.8 Despite this,
when an organism dies, cells rage against the process. “Cells don’t
want to die,” said Gulnaz Javan, a forensic scientist at Alabama State
University. Survival is programmed into their molecular makeup.
While the moment of clinical death is absolute, some cells
defy this moment. As postmortem time marches on, cells that
remain stable in low-nutrient and oxygen conditions, such
34 T H E SC I EN T I ST | the-scientist.com
as stem cells, survive. 9 During this period of cellular after-
life, these cells release molecular distress calls in an ongoing
display of death resistance. “Cells within tissues struggle to
survive by changing their transcriptional programs to cause
upregulation of developmental pathways,” Javan said.
Unwinding the clock
Javan coined the term thanatotranscriptome, which derives
from the Greek word for death, thanatos, to describe post-
mortem gene expression. Javan and her colleagues examined
gene expression in postmortem human liver tissue and found
a substantial increase in expression of a gene that promotes
cell survival known as X-linked inhibitor of apoptosis protein
(XIAP).10 They also found increased expression of XIAP and
other prosurvival genes such as BAG1 and BCL2 in human
prostate autopsy tissue.11
Peter Noble, adjunct professor of microbiology at the Univer-
sity of Alabama Birmingham, and his team examined gene tran-
scription in zebrafish and mice in the days after death and made
an unexpected discovery.5 “There was about one to two percent of
the total transcriptome that was active,” Noble said. This included
one thousand and sixty-three genes to be exact, some of which
became active up to two days after death. Other researchers also
discovered increased gene transcription after death in human tis-
sues, including brain, blood, and skin samples.12-16
Cells don’t want to die.
MODIFIED FROM © ISTOCK.COM, ILYA LUKICHEV, LUCKYSTEP48; DESIGNED BY ERIN LEMIEUX
—Gulnaz Javan, Alabama State University
Noble and his team categorized these zombie genes into
functional categories, including those that play a role in devel-
opment, cancer, stress responses, inflammation, immunity, cell
death, nutrient transport, and epigenetic processes. One of the
key developmental genes activated was hypoxia inducible fac-
tor (HIF), which is part of a group of transcription factors that
respond to low oxygen levels by regulating other oxygen sensi-
tive genes that are active in early embryonic development and
certain physiological and disease states.17 HIF transcription
factors regulate the expression of hundreds of genes through
various molecular signaling pathways that have far reaching
roles in cell proliferation, growth, metabolism, and survival.
Noble describes the zombie gene phenomenon as a genetic
unraveling of the developmental clock. “After death, all hell breaks
loose and just starts unwinding,” he said. The usual genetic and
epigenetic brakes that silence developmental genes throughout
an organism’s lifetime are released.
Beyond the veil
As researchers unravel the secrets encoded in zombie genes, they
also discover their far-reaching scientific relevance. “When we
GENE ACTIVITY
IN THE CELLULAR
AFTERLIFE
After an organism dies, most of its cells begin to extinguish
activity and die shortly afterward. However, other cells exhibit
a curious behavior. Instead of winding down their operations,
certain gene activities resurrect.

LOW OXYGEN BEGINNINGS

During the earliest stages of embryonic development, stem cells prolif-
erate in a low oxygen environment. The genes that drive this stage are
active for a short period of time. After the blastocyst implants into the
uterus, eventually, oxygen levels increase and gene activity that was
previously maintained by low oxygen levels silences.

SURVIVAL INSTINCT
Cellular life after death may seem paradoxical, but sudden changes in oxygen levels
trigger protective responses in cells. Genes that are transcribed during the low oxy-
gen phase of embryonic development are reactivated when oxygen levels plumet
after organismal death. Many emergency-mode genes also activate to support cell
survival, including those involved in inflammation, immunity, stress responses, and
cancer. Scientists studied this in zebrafish and mice, as well as human blood, pros-
tate, liver, and brain tissue samples.

REAL-WORLD ZOMBIE GENES

There are practical applications for understanding why and how genes are
activated after death. For example, forensic scientists apply insights from
postmortem gene transcription to estimate the time of death in criminal
cases. Scientists also use information about cancer gene reactivation to
improve organ transplant outcomes. Performing transplant surgery before
these genes become active may help reduce the high incidence of cancer
in organ transplant recipients.
 Gulnaz Javan is a forensic
scientist at Alabama State
University who studies the
practical applications of
zombie genes.
started this work, people
thought we were nuts. They
thought ‘who wants to
study death?’” Noble said.
“But it turns out that there
are many practical reasons
for doing so.” For example,
zombie gene expression is
being explored as a forensic
tool to predict the postmortem interval—the time between death
and the start of a criminal investigation—based on their precise and
time-sensitive expression.18
Thanatotranscriptome research also informs cancer and
organ transplant science. “When you transplant an organ, you're
taking it from a dead donor. In some cases, there's an increase
in cancer gene expression,” Noble said. This is important, given
that the incidence of cancer in organ transplant recipients is
significantly higher than the general population. “The common
theme is that there is an immunological problem, but when
you transfer a kidney or liver to a donor, the cancer genes have
already been turned on in the dead person, and it's being trans-
ferred to the recipient,” Noble explained.
Javan intends to further study postmortem decomposi-
tion of the prostate and liver, which are among the organs that
remain intact the longest after death, to inform organ transplant
research. “My team is assessing mRNA transcript abundance in
postmortem prostate and liver tissues to obtain the list of can-
didate genes that can be used in the development of test kits to
be used by organ transplantologists,” Javan said. Such biomark-
ers can improve the match between organ donors and recipients
and reduce the rate of transplant rejection.
Despite advances in understanding the thanatotranscrip-
tome, the cellular afterlife remains shrouded in mystery. “When
I was in college, I wondered what happens after we die. Does
every cell in our body die at the same time or is there life that
goes on?” Javan said. As scientists continue to unearth the
answer to this question, zombie genes may hold the molecu-
lar keys to understanding far-reaching processes in the human
body. The conserved molecular pathways that shape early
embryonic development and resurrect postmortem gene activ-
ity suggest a continuum along the thin strand that binds cellu-
lar life from the cradle to the grave. “We really don't know what
happens when an organism dies,” Noble said. How long certain
genes remain active and whether they might stay dormant for
protracted periods in cells that survive below the threshold of
oxygen and nutrient availability that currently defines the needs
of a living cell remains to be seen. In the meantime, scientists
36 T H E SC I EN T I ST | the-scientist.com
continue to unravel the truth one gene at a time in their quest
to uncover what lies beyond the veil. J
References
1. Fathollahipour S, et al. Oxygen regulation in development: lessons from
embryogenesis towards tissue engineering. Cells Tissues Organs. 2018;205(5-
6):350-371.
2. Michiels C. Physiological and pathological responses to hypoxia. Am J Pathol.
2004;164(6):1875-1882.
3. Larsen’s Human Embryology—5th Edition. Available online:
https://www.elsevier.com/books/larsens-human-embryology/
schoenwolf/978-1-4557-0684-6 (accessed on 6 October 2023).
4. Podkalicka P, et al. Hypoxia as a driving force of pluripotent stem cell
reprogramming and differentiation to endothelial cells. Biomolecules.
2020;10(12):1614.
5. Pozhitkov AE, et al. Tracing the dynamics of gene transcripts after organismal
death. Open Biol. 2017;7(1):160267.
6. Scott L, et al. Life and death: A systematic comparison of antemortem and
postmortem gene expression. Gene. 2020;731:144349.
7. Arya R, White K. Cell death in development: Signaling pathways and core
mechanisms. Semin Cell Dev Biol. 2015;39:12-19.
8. Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer.
Nature. 2001;411(6835):342-348.
9. Cieŋla J, Tomsia M. Cadaveric stem cells: their research potential and
limitations. Front Genet. 2021;12:798161.
10. Javan GT, et al. The apoptotic thanatotranscriptome associated with the liver
of cadavers. Forensic Sci Med Pathol. 2015;11(4):509-516.
11. Tolbert M, et al. The thanatotranscriptome: Gene expression of male
reproductive organs after death. Gene. 2018;675:191-196.
12. Dachet F, et al. Selective time-dependent changes in activity and cell-specific
gene expression in human postmortem brain. Sci Rep. 2021;11(1):6078.
13. Antiga LG, et al. Cell survival and DNA damage repair are promoted
in the human blood thanatotranscriptome shortly after death. Sci Rep.
2021;11(1):16585.
14. Ferreira PG, et al. The effects of death and post-mortem cold ischemia on
human tissue transcriptomes. Nat Commun. 2018;9(1):490.
15. Zhu Y, Wang L, Yin Y, Yang E. Systematic analysis of gene expression
patterns associated with postmortem interval in human tissues. Sci Rep.
2017;7(1):5435.
16. Abouhashem AS, et al. The prolonged terminal phase of human life
induces survival response in the skin transcriptome. Preprint. bioRxiv.
2023;2023.05.15.540715.
17. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell.
2012;148(3):399-408.
18. Hunter MC, et al. Accurate predictions of postmortem interval using
linear regression analyses of gene meter expression data. Forensic Sci Int.
2017;275:90-101.
GULNAZ JAVAN
 Present your paper in
The Scientist’s Journal Club.

Apply today with our simple application form to share your

cutting-edge research in The Scientist’s Journal Club.

DETAILS
• 15-minute journal club-style talks geared toward
an audience of life scientists
• Paper topic must be life science/molecular biology
• Paper must be less than six months old at the time
of application
APPLY TODAY!

SUBMISSION
DEADLINE

January 31,
2024
A Story
of FIRE
and Mice
Studying how microglia control myelin
growth and prevent its degeneration
helps scientists better understand and
address neurodegenerative diseases.

BY NIAMH McNAMARA, PhD AND

VERONIQUE MIRON, PhD
 D
espite decades of research, scientists still grapple with
the question of why neuronal health is compromised in
neurodegenerative diseases. For a long time, scientists
believed that Alzheimer’s disease resulted from toxic build-up of
the proteins amyloid and tau, which normally aid neural growth,
repair, and stability in the brain. Based on this idea, they devel-
oped therapies to break down these proteins, but those treat-
ments did not completely improve patients’ cognitive abilities
in clinical trials. This pushed scientists to explore other factors
beyond protein toxicity that damage neuronal health.
In 2004, George Bartzokis, a neuroscientist at the University
of California, Los Angeles, first hypothesized that myelin, the pro-
tective membrane that wraps around axons, might be the key to
understanding and treating Alzheimer’s disease.1 Myelin is essen-
tial for neuronal function. It provides axons with both the insula-
tion and the nutrients they need to survive and thrive. Myelin is
also central to efficient learning and memory encoding.2
In the early 2000s, researchers studied the brains of rhesus
monkeys that were older than 30 years of age (their average lifes-
pan is ~35 years) to better understand aging. They found that
myelin in the brains of aged nonhuman primates showed struc-
tural changes and breakdown. Strikingly, these changes corre-
© ISTOCK.COM, BAWANCH AND KUDRYAVTSEV PAVEL; CARSTEN DITTMAYER; NIAMH McNAMARA
lated with the degree of cognitive decline in the monkeys.3,4,5
Electron microscopy shows myelin overgrowth around axons in the white
matter of a patient with ALSP. Myelin destabilization and eventual degra-
dation are the hallmarks of neurogenerative disorders.
Dysregulated myelin in FIRE mice indicates the essential role that microg-
lia play in myelin stability.
When scientists conducted MRI studies in humans, they found
that myelin shows damage up to 20 years prior to Alzheimer’s dis-
ease onset.6 Together, these studies hinted that myelin damage is
intrinsically linked with cognitive decline and is an early event in
Alzheimer’s disease progression.
Despite this evidence, there were no follow up studies to val-
idate this hypothesis for almost two decades, partially because
researchers assumed that the myelin changes resulted from,
rather than caused, neuronal loss. But recently, researchers at
the Third Military Medical University and the University of Cal-
ifornia, San Francisco observed myelin degeneration coupled
with a high rate of myelin repair in a mouse model of Alzheim-
er’s disease. Although the high repair rate could not compensate
for the ongoing damage, they found that enhancing myelina-
tion improved cognition and neuronal function, irrespective of
the amount of amyloid buildup.6,7 In fact, scientists at the Max
Planck Institute for Multidisciplinary Sciences recently discov-
ered that myelin damage itself can drive amyloid accumulation
in mouse models of Alzheimer’s disease.8 These results finally
provided evidence supporting Bartzokis’ hypothesis. 9,10
The microglia and myelination connection
While these studies answered some questions, they raised several
more. For example, if researchers could determine why myelin
degenerates, they might be able to prevent dementia onset. The
first step towards studying myelin degradation in diseases was to
understand how myelin maintains its structure and function in
a healthy brain. For this investigation, researchers turned to an
internal partner: microglia.
Microglia are a subset of glial cells that support neurons and
keep the microenvironment in the central nervous system healthy
and intact. Acting as soldiers on the brain frontlines, microglia
function as immune cells, protecting the brain from pathogens,
WINTER 2023 | T H E S C IE N T IST 3 9
 MICROGLIA INFLUENCE MYELIN HEALTH
In FIRE mice, the lack of microglia causes myelin overgrowth and eventual degeneration,
indicating that microglia may contribute to age-related neurodegenerative diseases.
FIRE mice Microglia absence
injury, or any harmful triggers. In the last decade, researchers
have learned that microglia also contribute to healthy brain func-
tion. In fact, microglia consistently contribute to myelin health
throughout the lifespan.
Researchers recently found that young mice lacking microg-
lia had less myelin in their developing brains than mice with
intact microglia.11,12 This suggested that microglia mediated
myelination in the developing brain by driving the production
of oligodendrocytes, which produce myelin. However, there was
one key challenge with these studies. The tools available for elim-
inating microglia at the time also targeted another small pop-
ulation of central nervous system immune cells known as bor-
der-associated macrophages, which are found in the meninges,
surrounding blood vessels, and in areas that contact cerebrospi-
nal fluid. Scientists did not know the potential contributions of
these cells to myelination.
In 2019, researchers at the University of Edinburgh created a
game-changing new mouse model. By using CRISPR-Cas9, they
deleted the FIRE sequence, which is a super enhancer in the
gene encoding CSF1R, a receptor required for microglia devel-
opment and survival.13 FIRE has redundant function in border-
associated macrophages, so FIRE knockout mice (or FIRE mice)
lack microglia but retain border-associated macrophages. We
used this new model to unpack the specific roles of microglia in
myelination for the first time.
Microglia control myelin growth
In our lab at the University of Edinburgh, we eagerly investi-
gated myelin in these FIRE mice. We hypothesized that the lack
of microglia in the FIRE mice would cause deficient myelination
during early development, an idea in line with results from pre-
4 0 T H E SC I EN T I ST | the-scientist.com
Myelin overgrowth Demyelination
vious studies that used global macrophage depletion models. But
we were in for a surprise.
In our analysis, we found no deficit in myelin formation
in these mice. Perplexed, we spent months analyzing various
myelin-associated proteins and characterizing oligodendro-
cyte numbers, but they all appeared unaffected. Finally, when
we took a closer look at the ultrastructural level using elec-
tron microscopy, we had a breakthrough. We observed that
myelin was present in FIRE mice, but in unexpected abun-
dance. In the absence of microglia, young FIRE mouse brains
formed excess myelin during development that remained in
adult FIRE mice.
These findings suggested that microglia actively engage
in regulating myelin growth. To confirm our theory using
another approach, we depleted microglia and macrophages in
adult mice using the previous gold standard, a pharmacologi-
cal drug that inhibits CSF1R. We saw the same result: FIRE
mouse brains showed excess myelin formation after just one
month of treatment.
Next, we explored whether this phenomenon also occurred in
humans. We obtained precious tissue from a rare human neuro-
degenerative disease known as adult-onset leukoencephalopathy
with axonal spheroids and pigmented glia (ALSP) from Werner
Stenzel, a neuropathologist at Charité-Universitätsmedizin Ber-
lin. In patients suffering from ALSP, heterozygous mutations in
the CSF1R gene result in approximately half the normal amount
of microglia, specifically in myelin-enriched areas in the white
matter. Patients with ALSP typically present with early-onset
dementia and usually pass away in their 40s or 50s. In brain sam-
ples from patients with ALSP, we observed myelin overgrowth
similar to what we saw in FIRE mice.
 This revelation meant that microglia not only control myelin
formation during development, but they also regulate myelin
growth into adulthood. Moreover, myelin overgrowth when
microglia were absent or reduced in number looked remark-
ably similar to the disrupted myelin observed in the aged non-
human primates with cognitive decline. This suggested that in
the absence of healthy microglia, myelin may age prematurely
and affect cognition.
We next assessed cognitive function in the FIRE mice by run-
ning the Barnes Maze test. In this test, mice learn to locate an
escape hole in a maze over several trials. After a few days, we relo-
cate the escape chamber; how the mice adapt to the new situation
reflects their cognitive flexibility. The FIRE mice showed deficits
in cognitive flexibility, which is one of the first cognitive functions
that declines with age.14,15 Previous studies have also suggested
that the extent to which this ability is lost with age could predict
development of Alzheimer’s disease.16
Microglia prevent myelin degeneration
Since aging perturbs myelin structure, and myelin degenerates
in Alzheimer’s disease, we next wondered whether the myelin
structural changes seen in the absence of microglia rendered
ILLUSTRATION BY © ASHLEIGH CAMPSALL, ADAPTED FROM A GRAPHIC BY NIAMH MCNAMARA; © ISTOCK.COM, NAEBLYS
myelin vulnerable to degeneration. In samples from patients
with ALSP, we noticed some age-related differences in the
myelin. A younger patient who died from an unrelated cause
had abundant and extremely overgrown myelin. But in an older
patient, much of the myelin had degenerated; any remaining
myelin was overgrown.
This led us to investigate the role of microglia in myelin
maintenance. By six months of age in FIRE mice, we saw clear
evidence of myelin degeneration. In the absence of microglia,
myelin quickly went from overgrown to broken down. This was
a strange phenomenon, and mechanistically, it didn’t make
much sense.
To find out whether myelin broke down due to the prolonged
absence of microglia in FIRE mice, we pharmacologically depleted
microglia and macrophages for one month in five-month-old wild
type mice. We observed the same degenerated myelin in these
mice, indicating that the function of microglia in maintaining
myelin health is increasingly important as the brain ages.
Microglia burnout
Burnout is characterized by utter exhaustion due to excessive
workload and stress over a prolonged period of time. Microg-
lia may also suffer from burnout, and understanding how this
happens could be central to our fight against dementia. Numer-
ous sophisticated single-cell transcriptomic sequencing studies
have documented the appearance of a disease-associated microg-
lia population in aging and in several disease contexts, including
Alzheimer’s disease. This population may appear as a response to
the increasing demands of the aging brain. With too many tasks
to juggle, microglia may lose their ability to maintain myelin as
well as they did when the brain was younger.
If we could rejuvenate microglia to their younger selves, per-
haps we could prevent the degeneration of myelin and poten-
tially the development of neurodegenerative disease in the aging
brain. And if we can thwart this process, we might just be able
to help our loved ones preserve their memories until well into
their autumn years. J
Conflict of interest statement
Veronique E Miron has received consultancy or research funds
from Novartis, Biogen, GSK, Astex Pharmaceuticals, Clene Nano-
medicine, ReWind Therapeutics.
Niamh McNamara recently completed her PhD on microglial reg-
ulation of myelin integrity at the University of Edinburgh. She is
now pursuing postdoctoral research at the Netherlands Institute
for Neuroscience in Amsterdam.
Veronique Miron is a neuroimmunology professor leading research
laboratories at the University of Toronto and the University of
Edinburgh that investigate what regulates myelin health and
pathology across the lifespan.
References
1. Bartzokis G. Age-related myelin breakdown: A developmental model of
cognitive decline and alzheimer’s disease. Neurobiol. Aging. 2004;25(1):5-18.
2. Xin W and Chan JR. Myelin plasticity: Sculpting circuits in learning and
memory. Nat. Rev. Neurosci. 2020;21(12):682-694.
3. Peters A. The effects of normal aging on myelin and nerve fibers: a review.
J. Neurocytol. 2002;31(8/9):581-593.
4. Peters A. The effects of normal aging on myelinated nerve fibers in monkey
central nervous system. Front. Neuroanat. 2009;3.
5. Peters A and Sethares C. Aging and the myelinated fibers in prefrontal cortex and
corpus callosum of the monkey. J. Comp. Neurol. 2001;442(3):277-291.
6. d’Arbeloff T, et al. White matter hyperintensities are common in midlife and
already associated with cognitive decline. Brain commun. 2019;1(1).
7. Chen J-F, et al. Enhancing myelin renewal reverses cognitive dysfunction in a
murine model of alzheimer’s disease. Neuron. 2021;109(14).
8. Depp C, Sun T, et al. Myelin dysfunction drives amyloid-` deposition in models
of Alzheimer’s disease. Nature. 2023;618(7964):349-357.
9. Bartzokis G, et al. White Matter Structural Integrity in healthy aging adults and
patients with alzheimer disease. Arch. Neurol. 2003;60(3):393.
10. Graff-Radford J, et al. White matter hyperintensities: Relationship to amyloid
and tau burden. Brain. 2019;142(8):2483-2491.
11. Erblich B, et al. Absence of colony stimulation factor-1 receptor results in loss
of microglia, disrupted brain development and olfactory deficits. PLoS ONE.
2011;6(10).
12. Hagemeyer N, et al. Microglia contribute to normal myelinogenesis and to
oligodendrocyte progenitor maintenance during adulthood. Acta Neuropathol.
2017;134(3):441-458.
13. Rojo R, et al. Deletion of a Csf1r enhancer selectively impacts CSF1R expression
and development of tissue macrophage populations. Nat. Commun. 2019;10(1).
14. Boone KB, et al. Wisconsin card sorting test performance in healthy, older adults:
Relationship to age, sex, education, and IQ. J. Clin. Psychol. 1993;49(1):54-60.
15. Moore TL, et al. Executive system dysfunction occurs as early as middle-age in
the rhesus monkey. Neurobiol. Aging. 2006;27(10):1484-1493.
16. Ballesteros S, et al. Cognitive function in normal aging and in older adults with
mild cognitive impairment. Psicothema. 2013;25(1):18-24.
WINTER 2023 | T H E S C IE N T IST 41
 The Literature
Rebranding Mitochondria
As scientists realize the multifaceted role of mitochondria, some
feel that the “powerhouse of the cell” analogy is out of date.
BY DANIELLE GERHARD, PhD
I
n a popular Indian parable, a few blind
men interact with an elephant for the
first time and imagine what it looks like.
The man touching the tusk may describe
the elephant as a spear, while the person
tugging the tail may think that it’s a rope.
All of them miss the big picture. The moral
of the story is that narrow experiences can
advance inaccurate perspectives.
Martin Picard, a mitochondrial biol-
ogist at Columbia University, likened
mitochondria to the elephant in the
fable. “Mitochondria are diverse,” said
Picard. “To some people, that’s a gentle
reminder. For some people, that’s an eye-
opening claim.”
Mitochondria constantly churn out
chemical energy to fuel the extensive net-
work of biochemical reactions occurring
throughout the cell, thus inspiring the
universal aphorism of the “powerhouses
of the cell.” However, the last few decades
have ushered in a more nuanced under-
standing of the organelle with growing
With a greater understanding of the many roles of mitochon-
dria, the more precise you can be and the better and clearer
the hypothesis you’ll come up with will be.
evidence that it contributes to a num-
ber of diseases, regulates several cellu-
lar processes, and plays multifaceted
roles in cells. This attracted many scien-
tists, even those who may have a narrow
4 2 T H E SC I ENTI ST | the-scientist.com
understanding of the organelle, to mito-
chondrial biology.
Considering this, Picard spearheaded
two perspectives that he hopes will serve
as an invaluable compendium on the
organelle for experts and visitors to the
Mitochondria function like cellular processors, like little anten-
nas that can receive information, integrate information, and then
produce signals that influence the cell and the whole organism.
field alike.1,2 In the first perspective, pub-
lished in Cell Metabolism, Picard and
Orian Shirihai, a mitochondrial biologist
at the University of California, Los Ange-
les, made the case that the powerhouse
analogy is dated; they instead focus on
the organelle as the great communica-
tor of the cell.1 “Mitochondria function
like cellular processors, like little anten-
—Mike Murphy, University of Cambridge
nas that can receive information, inte-
grate information, and then produce sig-
nals that influence the cell and the whole
organism,” said Picard. Inputs include
hormones, metabolites, and nutrients
that direct output signals that orches-
trate metabolic pathways, gene expres-
sion, and drive adaptive behaviors.
Although Picard and his colleagues
rebranded the organelle under the
umbrella of communicators, they empha-
—Martin Picard, Columbia University
sized that context matters. Early in devel-
opment, mitochondria diversify and spe-
cialize as different cell types and tissues
emerge. Just as scientists continue to dis-
cover and define functionally and molec-
ularly distinct cell types, Picard noted
rising evidence of mitochondrial phe-
notypes, or mitotypes, that likely influ-
ence signal processing and mitochondrial
communication.3 For example, Picard’s
team and others showed that brain cells
in mice exhibit regional and cell-spe-
cific functional differences, while human
immune cells vary in ATP production and
mitochondrial DNA copy number.3-5
“Now that we know this, we need a
decent nomenclature system that will
allow us to teach the next generation to
formulate specific hypotheses and then
design research to test that with a certain
level of specificity,” said Picard. In their
Nature Metabolism perspective, Picard
and his colleagues proposed a terminol-
 Mitochondria vary in their cell-dependent
characteristics, such as topology and DNA
copy number.
Mitochondria are diverse
and multifaceted, and
they exhibit a high
degree of heterogeneity Mitochondrial morphology and
across tissues. molecular composition, including
their lipid, protein, and RNA profiles
can differ even within the same cell.

Mitochondria undergo a range

of cellular activities, including
protein synthesis, metabolite and
ion transport, and DNA repair.

ATP
synthase Mitochondria use a variety
of functions, such as lipid
synthesis, steroidogenesis,
ADP ATP and energy production. Dynamic interplay across
these domains allows
Endoplasmic
reticulum these shape-shifting
organelles to respond to
their environments. In
Mitochondria engage in different behaviors, like this evolving framework,
motility, fission and fusion, and communication mitochondria are viewed
with other mitochondria and organelles. as cellular processors.

ogy system to increase specificity in the
language of mitochondrial science.2 Their
system distinguishes between the multi-
tude of cell-dependent properties, molec-
ular features, activities, functions, and
behaviors employed by mitochondria.
Mike Murphy, a mitochondrial biolo-
gist at the University of Cambridge who
was not involved with writing the per-
DESIGNED BY ASHLEIGH CAMPSALL
signaling. “With a greater understanding
of the many roles of mitochondria, the
more precise you can be and the better
and clearer the hypothesis you’ll come up
with will be,” said Murphy.
“I’m supportive of the goal, [but] I’m reluc-
tant to go along with a rigid nomenclature,”
said Murphy. Mitochondria are dynamic and
constantly adapting in response to a changing
ago, when people realized ‘ooh, we’re
made of cells,’” said Picard. J
References
1. Picard M, Shirihai OS. Mitochondrial signal
transduction. Cell Metab. 2022;34(11):1620-1653.
2. Monzel AS, et al. Multifaceted mitochondria:
Moving mitochondrial science beyond function
and dysfunction. Nat Metab. 2023;5(4):546-562.

3. Rausser S, et al. Mitochondrial phenotypes in

spectives, agreed with Picard’s call for
more precise language. “We’re using
vague terms like mitochondrial dysfunc-
tion, and it’s not clear what that means,”
said Murphy. Instead, descriptions
should focus on the specific process that
has gone awry, such as calcium homeo-
stasis, oxidative phosphorylation pro-
ducing ATP, or contributions to immune
environment, which could make it difficult to
pigeonhole these shapeshifting organelles into
one classification over another.
Whether scientists adopt the pro-
posed terminology system remains to be
seen, but appreciation of the organelles’
incredible diversity is only growing. “In
the world of mitochondrial biology, we’re
in the same place as probably 200 years
purified human immune cell subtypes and cell
mixtures. eLife. 2021;10:e70899.
4. Rosenberg A, et al. Brain mitochondrial diversity
and network organization predict anxiety-
like behavior in male mice. Nat Commun.
2023;14:4726.
5. Fecher C, et al. Cell-type-specific profiling
of brain mitochondria reveals functional
and molecular diversity. Nat Neurosci.
2019;22:1731-1742.

WINTER 2 02 3 | T H E S C IE N T IST 4 3
 THE LITERATURE
Biosensors for Colorectal Cancer
Engineered bacteria sound the alarm on a common oncogenic mutation.
BY HANNAH THOMASY, PhD
T
he human gut is awash in a sea
of microbes that quietly ferment
fibers, produce vitamins, and
exchange information with the immune
system.1 Now, scientists are tasking bac-
teria with yet another job as they spelunk
their way through the digestive system:
cancer detection.
An international team of researchers
engineered a bacterial biosensor capa-
ble of identifying a cancer-associated
DNA mutation, which they published in
the journal Science.2 The research team
included molecular biologists Robert
Cooper and Jeff Hasty of the University
of California, San Diego and bowel can-
cer researchers Josephine Wright and
Susan Woods at the South Australian
Health and Medical Research Institute,
4 4 T H E SC I EN T I ST | the-scientist.com
and Daniel Worthley at the Colonoscopy
Clinic. The study authors hope that this
technology will one day aid in the early
diagnosis of colorectal cancer, one of the
most common causes of cancer-related
death globally.
While scientists have previously engi-
neered bacteria to detect inflammation or
bleeding in the gut, this is the first bacte-
rial biosensor that detects a specific DNA
sequence from host tissues. To accomplish
this feat, scientists leveraged Acineto-
bacter baylyi’s ability to take up extracel-
lular DNA and integrate these sequences
into its own genome.
“Using living bacteria to sense things
in the gut and detect disease is some-
thing that I find very exciting,” said David
Riglar, a microbiome researcher at Impe-
Globally, colorectal cancer is the third most
common cancer; bacterial biosensors may one
day help with early detection of this disease.
rial College London who was not involved
in the study. “Taking these naturally com-
petent bacteria, detecting DNA changes,
and then using that as a biosensor is a
really cool advance.”
In this study, researchers wanted to
engineer A. baylyi to detect a common
© ISTOCK.COM, LUISMMOLINA
colorectal cancer marker: a mutation in
codon 12 of the KRAS gene. “At the time,
it seemed like a fairly far-fetched idea,”
recalled Worthley. By bringing together
an interdisciplinary team with expertise
in synthetic biology and animal models of
colorectal cancer, the researchers achieved
this lofty goal.
 In their early proof-of-concept exper-
iments, the researchers genetically tin-
kered with both A. baylyi and the tumor
organoids that they wanted the bacteria
to detect. They engineered tumor cells
with a functional copy of the antibiotic
Taking these naturally
competent bacteria, detecting
DNA changes, and then using
that as a biosensor is a
really cool advance.
—David Riglar, Imperial College London
resistance gene, kanR, flanked by KRAS
homology arms. The bacteria had match-
ing KRAS homology arms, plus two stop
codons that prevented the expression of
kanR. When the bacteria gobbled up the
donor tumor DNA, the homology arms
aligned the DNA sequences and the bac-
teria integrated the functional kanR into
their own genomes, enabling them to
grow on antibiotic-laced plates.
To create bacteria that specifically
detected mutant KRAS, the researchers
harnessed the bacteria’s own CRISPR-Cas
machinery, directing these molecular scis-
sors to chop up wild-type, but not mutant
KRAS. This would kill any bacteria that
acquired the wild-type KRAS.
The researchers then tested these bac-
teria against colorectal cancer organoids
with and without the engineered donor
DNA. Only the bacteria cocultured with
the engineered tumors acquired antibiotic
resistance, showing that the sensor bac-
teria could discriminate between normal
and donor tumors.
Next, the researchers tested the bio-
sensors in vivo by administering the
bacteria via enema to three groups:
mice without tumors, mice with nor-
mal colorectal tumors, and mice with
the engineered colorectal tumors. Again,
only the biosensors administered to the
mice with the engineered tumors grew in
the presence of the antibiotic, confirm-
ing that the bacteria could be used to sig-
nal the presence of engineered colorectal
cancer in mice.
While these data were promising,
human colorectal tumors don’t come engi-
neered with a perfectly placed antibiotic
resistance gene for bacteria to acquire. So,
the researchers adjusted their strategy to
detect natural tumor DNA with the KRAS
mutation. This time, they placed a repres-
sor gene inside the KRAS homology arms.
This gene prevented the expression of a
downstream kanR gene.
When the bacteria swapped their
KRAS DNA for the tumor’s KRAS, the
repressor was lost, allowing the antibi-
otic resistance gene to be expressed. As
before, wild type KRAS was targeted
for destruction by the CRISPR-Cas sys-
tem. In vitro, these new biosensors dis-
criminated between mutant and normal
KRAS by surviving and becoming antibi-
otic resistant only in the presence of the
cancer-associated mutation. The team
named this technique CATCH for cellu-
lar assay for targeted, CRISPR-discrimi-
nated horizontal gene transfer.
Since we’ve engineered all the sophistication within the cell,
we don’t need such a sophisticated laboratory outside the cell.
Despite these preliminary successes,
Riglar urged caution. “It’s important not
to run too far ahead in terms of thinking
that these systems are ready to go into the
clinic,” he said.
“This is absolutely not the endpoint,”
Worthley agreed.
The researchers are currently working
on strategies to improve the biosensor’s
sensitivity to natural tumor DNA in the
complex environment of the colon. Due to
concerns about administering antibiotic-
resistant bacteria to humans, they are also
developing other ways for the biosensors
to signal the presence of mutant KRAS.
To be commercially viable, the biosensor
bacteria must be delivered orally, mean-
ing that they will need to survive their
journeys through the digestive system
and be able to report their findings on
the other side.
Eventually, however, Worthley hopes
that these biosensor bacteria will one
day be used as point-of-care diagnos-
tics in remote or low-resource areas
such as the Australian outback. “Since
we’ve engineered all the sophistica-
tion within the cell, we don’t need such
a sophisticated laboratory outside the
cell,” he said.
Researchers hope for broader appli-
cations as well. Instead of turning on
an antibiotic resistance gene when they
sense tumor DNA, for example, the bac-
teria could be engineered to turn on pro-
duction of a genotype-specific small-mol-
ecule therapeutic, delivering treatment
precisely where it’s needed. Bacteria could
be engineered to detect and respond
appropriately to various oncogenic muta-
tions, or even difficult-to-treat infections
like Clostridium difficile. Worthley sees
this potential to marry diagnosis and
—Daniel Worthley, Colonoscopy Clinic
therapy as the major advantage of these
engineered bacteria. J
Conflict of interest statement:
J.H., D.W., and S.W. have equity in Gen-
Cirq Inc., which focuses on cancer thera-
peutics. D.W., J.H., R.C., S.W., and J.W.
have a provisional patent application on
this technology.
References
1. Bull MJ, Plummer NT. Part 1: The Human Gut
Microbiome in Health and Disease. Integr Med
(Encinitas). 2014;13(6):17-22.
2. Cooper RM et al. Engineered bacteria detect
tumor DNA. Science. 2023;381(6658):682-686.
WINTER 2 02 3 | T H E S C IE N T IST 4 5
 THE LITERATURE

Tracking Down Innate Immune Cells in Multiple Sclerosis

A novel PET tracer targeting a receptor in myeloid cells can help monitor disease progression in a
mouse model of multiple sclerosis.

BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

M
ultiple sclerosis (MS) is a
debilitating disease in which
a patient’s immune system
attacks the myelin protective coating
around nerve cells, causing inflamma-
tion and disrupting communication to
and from the brain. Evidence indicates
that myeloid cells, components of the
innate immune system, aid the initia-
tion, progression, and remission of MS.1
However, scientists lacked methods to
specifically track down myeloid cells as
they turn from friend to foe and mount a
more damaging immune response.
To fill this gap, researchers led by
Michelle James, a radiochemist and
neuropharmacologist at Stanford Uni-
versity, developed a novel PET tracer
that targets the triggering receptor
expressed on myeloid cells 1 (TREM1).
They showed that in vivo TREM1 PET
imaging allowed early disease detection
and treatment monitoring in a mouse
model of MS.2 Their findings, published
in Science Translational Medicine, posit
that TREM1 is an early marker of mal-
adaptive innate immune responses, and
highlight the biomarker’s potential use
for monitoring disease progression and
response to treatment in patients with
the disease.
“We have a way of lighting up where
inflammation is in the whole body and
brain in the context of MS,” said James,
whose work focuses on the development
of imaging agents for visualizing neuroim-
mune interactions. “We have never been
able to do that before with such specificity.”
James did not initially focus on
TREM1. The membrane receptor caught
her attention when she was looking at a
transcriptomic data set from her colleague
Katrin Andreasson, a neurologist at Stan-
ford University and coauthor of the study.
The pair noticed that the expression of
TREM1 was upregulated only when there
was a more harmful immune response.
“We thought, ‘wow, this could be a really
great biomarker to tell us when there is
something really bad occurring with the
innate immune system in the context of
diseases,’” James recalled.
To test this idea, the researchers
focused on MS and used the experi-
mental autoimmune encephalomyeli-
tis (EAE) mouse model, which recapit-
ulates important aspects of the disease
such as muscle weakness and changes
in the innate immune response. They PET imaging of TREM1 enables detection
developed a PET tracer by radiolabel- of infiltrating proinflammatory myeloid cells
ing an anti-TREM1 antibody to specifi- in early stage disease in a mouse model of
multiple sclerosis.
cally track down TREM1+ cells and used
a PET imaging scanner to monitor the
movement of myeloid cells through the of the disease, when mice showed few
animal’s body as disease progressed. signs of loss of muscle function. “We liter-
TREM1 was selectively expressed ally did very excited happy dances in the
on peripheral myeloid cells in the EAE preclinical imaging facility because the
mouse model, and the researchers clarity of the images was just outstand-
observed central nervous system infiltra- ing. For PET imaging, it is kind of rare
tion of TREM1+ cells even in early stages to be able to see such clear-cut images
where you do not even need quantifica-
JAMES LAB, STANFORD UNIVERSITY

tion to see what is going on,” said Ais-

ling Chaney, a neuroimaging biologist at
We literally did very excited happy dances in the preclinical Washington University and coauthor of
imaging facility because the clarity of the images was just the study.
The team also found that TREM1
outstanding. For PET imaging, it is kind of rare to be able
PET signal showed higher sensitivity in
to see such clear-cut images where you do not even need detecting myeloid cell infiltration in the
quantification to see what is going on. central nervous system of EAE mice than
—Aisling Chaney, Washington University. the current gold standard PET tracer,

4 6 T H E SC I EN T I ST | the-scientist.com
which is widely used to detect neuroin-
flammation in vivo.
Given the specificity of TREM1 to
track harmful innate immune responses,
the team next investigated if it could be
used to indicate therapeutic response.
They treated EAE-induced mice with the
drug Siponimod and found a reduction
in TREM1 PET signal in drug-treated
mice. According to Chaney, these find-
ings reveal that TREM1 could be used
as a tool for screening different types of
therapies, even those that do not directly
target myeloid cells.
Using TREM1 knockout animals as
controls in the imaging experiments also
revealed a biological role for TREM1,
Chaney explained. “In the TREM1
knockout animals that we induced EAE
in, 50 percent of them just did not get
sick or they just did not get sick at the
same time point that we were looking
at in the wild type animals,” she said.
The team confirmed these observations
by pharmacologically blocking TREM1
with LP17, a peptide decoy receptor
known to attenuate TREM1 signaling,
and observed reduced disease severity
in EAE mice, suggesting therapeutic
potential for targeting TREM1.
To establish TREM1’s clinical rel-
evance, the researchers looked at brain
biopsy samples from two patients with
MS and examined the presence of
TREM1+ cells. Since patients would ben-
efit the most by detecting and diagnos-
ing MS at early stages, the team looked
for treatment-naïve, early-stage sam-
ples, which are not easy to obtain, James
explained. The team found a high num-
ber of TREM1+ immune cells in MS brain
lesions compared to non-MS samples,
indicating that TREM1 could be used to
monitor disease progression in humans.
The combination of multiple tech-
niques strengthened the researchers’
SMART GATEWAYS INTO THE LAB OF THE FUTURE
In this episode, Deanna MacNeil from The Scientist’s Creative Services
Team spoke with Sofie Salama and David Haussler, professors at the
University of California, Santa Cruz, to learn more about the smart
technology behind growing brain organoids.
SOFIE SALAMA, PhD
University of California, Santa Cruz
DAVID HAUSSLER, PhD
University of California, Santa Cruz
LISTEN HERE
findings, pointed out Daniele de Paula
Faria, a molecular imaging researcher
at the University of Sao Paulo, who was
not involved in the research. “This is a
complete study with promising results,”
she added.
James and Chaney plan to continue
exploring TREM1’s role in neurological
disorders. “TREM1 had actually not been
looked at in a lot of neurological disorders
before,” Chaney said. “It is opening up a
whole area of peripheral immune cells
and their implications in neurodegenera-
tive disorders.” J
References
1. Mishra MK, Yong VW. Myeloid cells - targets of
medication in multiple sclerosis. Nat Rev Neurol.
2016;12(9):539-551.
2. Chaney AM, et al. PET imaging of TREM1
identifies CNS-infiltrating myeloid cells in a
mouse model of multiple sclerosis. Sci Transl
Med. 2023;15(702):eabm6267.
 PROFILE

A Microbial Link to Parkinson’s Disease

Haydeh Payami helped uncover the genetic basis of Parkinson’s disease. Now, she hopes to
find new ways to treat the disease by studying the gut microbiome.

BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

A
t the age of 19, Haydeh Payami, now a geneticist at the Uni-
versity of Alabama, Birmingham, left Iran and came to the
United States with two suitcases and a fascination with genet-
ics. In the following years, she pursued a doctoral degree in genetics
and trailblazed her path as a researcher. Today, Payami leads a team
of motivated researchers and combines her love of genetics with the
microbial world to find ways to prevent and treat Parkinson’s disease.
“Haydeh has certainly been a pioneer,” said Sarkis Mazmanian,
a gut microbiome researcher at the California Institute of Technol-
ogy, who believes that Payami’s work contributed greatly to instill-
ing confidence in the idea that changes in the gut microbiome are
not a secondary consequence of Parkinson’s disease, but may actu-
ally contribute to it.
Payami’s interest in Parkinson’s disease did not begin with
the microscopic inhabitants of the human gut. It started with a
genetic investigation.

A genetic component of Parkinson’s disease?

In the early 1990s, Payami led a group at the Oregon Health and
Sciences University that studied the genetic basis of Alzheimer’s
disease. One day, an undergraduate student approached her with
the idea of investigating the genetics of Parkinson’s disease. “I
said, ‘Well, that’s fine. But choose either Parkinson’s or genetics
because Parkinson’s doesn’t have a genetic component,’” Payami
recalled. “This was 1992. Everybody was convinced that Parkin-
son’s was purely environmental.”
At that time, the only well accepted case of a genetic link
to Parkinson’s disease was an Italian family where individ-
uals from different generations suffered from the disease.1
Motivated by the student’s determination to pursue the idea
and the lack of knowledge about the role of genetics in Par-
kinson’s disease beyond that case, Payami and her colleagues
conducted a family study to detect the presence of familial
aggregation for the disease.
Haydeh Payami studies the interaction of the human genome, gut microbiome,
and environmental factors in causation, progression, and treatment of
Parkinson’s disease.
The reactions were mixed, she recalled. While some scien-
tists resisted the idea that genes play a role in cases of Parkin-
son’s disease with unknown causes, others were more open and
embraced the new discovery.
Since the mid-1990s, Payami and others have linked 90 risk
loci to Parkinson’s disease,3 but these genes still did not explain
the whole story. So, Payami turned her attention to investigating
how environmental factors and their interactions with genes influ-
ence the disease. In one study, for instance, her team conducted a
genome wide association study (GWAS) to investigate how genes
influence the protective effects of coffee in Parkinson’s disease.
UNIVERSITY OF ALABAMA, BIRMINGHAM

The team looked at the family histories, including individ-
ual records of illnesses and health conditions along with records
of parents and siblings of patients with Parkinson’s disease.
Spouses or friends served as controls to help identify character-
istic symptoms, including tremors, rigidity, and disease diag-
noses. First-degree relatives of patients showed an increased
risk for developing Parkinson’s disease, as indicated by a higher
number of patients with a parent or sibling with symptoms or
a diagnosis of the brain disorder.2 “We published that paper in
1994 and braced for the world to come down on us,” Payami said.
They found that heavy coffee drinkers who carried a variant of the
gene GRIN2A, which encodes a glutamate receptor involved in
excitatory transmission in the brain, had a lower risk for develop-
ing Parkinson’s disease.4
Although her findings added to other evidence that the envi-
ronment influences this neurodegenerative disorder, a gene-envi-
ronment interaction still did not provide all of the pieces of the
puzzle. “What else is there if it’s not genes and environment?” Pay-
ami puzzled. Then, the boom in microbiome research in the 2010s
gave her a new clue.

4 8 T H E SC I EN T I ST | the-scientist.com
Haydeh has certainly been a pioneer.
—Sarkis Mazmanian, California Institute of Technology.
Gut microbes in action
The study of the microbiota traces back to the 17th century, when
Antonie van Leeuwenhoek used his handmade microscopes to
describe the animalcules he found in people’s mouths and stools.
However, in the last 20 years, the human-associated microbiota
research area has experienced a surge, as scientists have developed
novel -omics approaches and bioinformatics tools to understand
the impact and influence of these microscopic beings on the human
body, including on the brain.
In the mid-2010s, Mazmanian’s team uncovered a connection
between the gut microbiome and hallmarks of Parkinson’s disease
in a mouse model of the disease.5 In human studies, however, the
association was less clear, and inconsistencies between studies
made the link difficult to establish.
“It was kind of a Wild West of a research area,” said Zachary
Wallen, a medical bioinformatician at Labcorp Oncology and a
former student of Payami’s. “There was no standardization of any-
thing; there were no guidelines to go by. So, we just had to pave
our own way to figure out how to tag the microbiome to Parkin-
son’s disease in the most robust way possible.”
Payami’s team set out to determine if an imbalance, or dysbiosis,
of the gut microbiome was present in patients with Parkinson’s dis-
ease. They collected stool samples from patients and neurologically
healthy individuals, extracted DNA, and sequenced the 16S rRNA to
determine the microbial composition. The team’s initial results pro-
vided evidence of an imbalance in the gut microbes of patients, uncov-
ering an abundance of some bacterial taxa and a reduction in others.6
Next, the team characterized gut dysbiosis in Parkinson’s dis-
ease in more detail by using more advanced bioinformatics tools
in their microbiome-wide association studies, which they mod-
eled after the rigor and standards of GWAS. They identified an
overabundance of opportunistic pathogens, bacteria that are nor-
mally harmless but can cause infections in immunocompromised
individuals, in the microbiomes of patients with Parkinson’s dis-
ease.7 Although this was the first time that a group of potentially
pathogenic microbes was found in samples from patients with
this neurodegenerative disorder, the idea that a pathogen could
trigger the disease was not entirely new.
In 2003, the anatomist Heiko Braak and his colleagues pro-
posed that nonfamilial forms of the disease could be initiated in the
gut by an unknown pathogen.8 After entering the body through the
nasal cavity and reaching the gut, this pathogen triggered the for-
mation of abnormal aggregations of the alpha-synuclein protein, a
hallmark of Parkinson’s disease. These aggregates then spread from
the periphery to the brain via the vagal nerve and olfactory tract,
causing progressive brain cell dysfunction and loss.
The identification of these opportunistic pathogens is one of
Payami’s major contributions to the field according to Wallen.
It is still uncertain whether any of them is the disease trigger-
ing pathogen that Braak referred to, but revealing their identi-
ties will allow researchers to test their specific involvement in
Parkinson’s disease.
More recently, Payami’s group used a metagenomic approach
instead of 16S rRNA sequencing to take a deeper look at the Parkin-
son’s disease gut microbiome. In this large-scale study, which included
490 patients with Parkinson’s disease and 234 neurologically healthy
controls, they confirmed previous findings of an overabundance of
opportunistic pathogens and uncovered microbial genes and pathways
that may contribute to Parkinson’s disease pathology, such as dysregu-
lation of the production and metabolism of neuroactive molecules and
lower levels of anti-inflammatory molecules.9
Wallen, who spent his graduate and postdoctoral years under
Payami’s supervision, remembers Payami’s determination to
produce the best science and her collaborative nature the most.
“She had an office; she hardly ever used it. We would just sit in
the middle of our lab and talk because she just wanted to con-
stantly talk and communicate,” he recalled.
According to Mazmanian, human studies like those conducted
by Payami’s team could provide clues to potential interventions.
For example, the opportunistic pathogens identified by her team
could be targeted and potentially removed from an individual
suffering from Parkinson’s disease. Additionally, supplementing
patients with the beneficial anti-inflammatory bacteria they lack
might also prove to be a viable strategy, he said.
Even though Payami’s main research goal is still to find a cure
for Parkinson’s disease, she hopes that by looking at the microbi-
ome, scientists can develop strategies to treat it.
“There are so many people who are suffering with this disease.
They’re not at an early stage, and they’re being ignored as far as
clinical trials,” said Payami. “Their microbiomes are sick. We may
not change their Parkinson’s, but we might be able to give them
some relief or maybe even slow disease progression.” J
References
1. Golbe LI, et al. A large kindred with autosomal dominant Parkinson’s disease.
Ann Neurol. 1990;27(3):276-282.
2. Payami H, et al. Increased risk of Parkinson’s disease in parents and siblings of
patients. Ann Neurol. 1994;36(4):659-661.
3. Bandres-Ciga S, et al. Genetics of Parkinson’s disease: An introspection of its
journey towards precision medicine. Neurobiol Dis. 2020;137:104782.
4. Hamza TH, et al. Genome-wide gene-environment study identifies glutamate
receptor gene GRIN2A as a Parkinson’s disease modifier gene via interaction
with coffee. PLoS Genet. 2011;7(8):e1002237.
5. Sampson TR, et al. Gut Microbiota Regulate Motor Deficits and Neuroinflammation
in a Model of Parkinson’s Disease. Cell. 2016;167(6):1469-1480.e12.
6. Hill-Burns EM, et al. Parkinson’s disease and Parkinson’s disease medications have
distinct signatures of the gut microbiome. Mov Disord. 2017;32(5):739-749.
7. Wallen ZD, et al. Characterizing dysbiosis of gut microbiome in PD: evidence for
overabundance of opportunistic pathogens. NPJ Parkinsons Dis. 2020;6:11.
8. Braak H, et al. Idiopathic Parkinson’s disease: possible routes by which
vulnerable neuronal types may be subject to neuroinvasion by an unknown
pathogen. J Neural Transm (Vienna). 2003;110(5):517-536.
9. Wallen ZD, et al. Metagenomics of Parkinson’s disease implicates the gut
microbiome in multiple disease mechanisms. Nat Commun. 2022;13(1):6958.
WINTER 2 02 3 | T H E S C IE N T IST 49
 PROFILE

A Journey With Metabolism, Parasites,

and Cancer
Piet Borst led stellar work on cell organelles, trypanosomes, and cancer drug resistance during
the golden age of biology.

BY LAURA TRAN, PhD

F
or Piet Borst, a physician-biochemist and molecular biolo- For more than five decades, Piet Borst profoundly influenced discoveries in
gist at the Netherlands Cancer Institute, success in science numerous fields and championed funding for science.
is a matter of luck. He described his career using the words
“luck” and “accident,” but his research was linked by a common
thread: scientific interest. Over 50 years, he passionately pursued While waiting for his clinical internship, he stumbled upon an
NETHERLANDS CANCER INSTITUTE

opportunities that led him to make seminal discoveries in mito-
chondria, parasitology, and oncology.
Mitochondria and trypanosomes
From an early age, Borst’s home was filled with discussions of
scientific results and collaborators. His father was a professor
of internal medicine and a clinical researcher. Following in his
father’s footsteps, Borst studied medicine at the University of
Amsterdam with the intent to pursue clinical research.
opportunity to conduct research in the biochemistry department
with Bill Slater, an enterprising biochemist at the University of
Amsterdam. “The Slater lab was fantastic,” Borst recalled. In fact,
he was so impressed with Slater as a supervisor and researcher that
when Slater later requested Borst’s support on a project, Borst hap-
pily accepted, despite being six months into his medical internship.
Pausing his internship, Borst spent the next three years pursuing
a PhD degree in Slater’s lab. At that time, he worked on mitochon-
drial properties in cancer cells and went on to uncover a metabolic

5 0 T H E SC I EN T I ST | the-scientist.com
cycle involving the mitochondria: the malate-aspartate shuttle,
which helps cells extract energy from sugar.1
After he completed his internships, Borst wanted to
become an endocrinologist. Because there was limited space
at the endocrinology clinic, he had to wait two years before
he could continue his training. So, Borst went to New York
University School of Medicine and joined the Nobel laureate
Severo Ochoa’s group as a postdoctoral researcher to study
RNA phage replication in Escherichia coli.2
Slater offered Borst a chair position back at the University
of Amsterdam. There, he combined mitochondria and nucleic
acids and discovered circular mitochondrial DNA in vertebrates
and in yeast.3 Borst noted that this circular DNA contained very
little genetic information, and that all of the mitochondrial pro-
teins must be encoded in nuclear genes and made in the cytosol
before being imported into the mitochondria.4
If you want to discover new biology, it
means that you cannot only do the obvious
experiments. You must sometimes be a little
more ingenious than the next person. So that
means that you sometimes tackle projects
that nobody has much faith in, and those
projects often lead to real discoveries.
—Piet Borst, Netherlands Cancer Institute
While working on mitochondrial DNA, he became interested
in the unusual mitochondrial DNA of African trypanosomes,
which cause sleeping sickness. Some of his most notable work
was the discovery of an organelle called the glycosome, which
contains glycolytic enzymes and his collaboration with parasi-
tologist George Cross, now at the Rockefeller University, to elu-
cidate the mechanism for antigenic variation in trypanosomes.5,6
Antigenic variation is a survival mechanism for trypanosomes
that periodically alters the variant surface glycoproteins to evade
the host immune response. Borst’s findings also elucidated a
DNA transposition mechanism for antigenic variation and dem-
onstrated trans-splicing as an essential step in synthesis of try-
panosome mRNA.7,8
While working on antigenic variation, Borst investigated
telomeres on chromosome ends of trypanosomes and found
a repeated sequence that was later also identified in human
telomeres.9 He also discovered an unusual base in trypano-
some DNA called base J (named after his graduate student,
Janet Gommers-Ampt, at the Netherlands Cancer Institute)
and described its biosynthesis and function.10 Base J is a modi-
fied version of thymine and plays a role in terminating growing
RNA chains in trypanosomes and similar parasites.11
Molecular pumps
In 1983, Borst moved to the Netherlands Cancer Institute. As
a physician, Borst was always interested in medical applica-
tions, so he started investigating genes involved with multi-
drug resistance (MDR) in cancer. He helped decipher the phys-
iologic functions of several ABC transporters, which sit in cell
membranes and transport compounds out of the cell to con-
tribute to MDR.
One of his notable discoveries was ABCB4, a phosphati-
dylcholine transferase that is essential for making bile.12 This
was an unexpected discovery because at the time, scientists
thought that bile salts passively extracted phosphatidylcholine
into bile. Borst found that ABCB4 transports phospholipids
actively into bile, and that the dysfunction of this transporter
gene led to severe liver disease.13
Further studies demonstrated ABCB4’s ability to also
transport some drugs.14 Borst discovered that one member
of the P-glycoprotein family prevents entry of amphipathic
toxins in the gut and the brain, which prevented certain oral
medications from being absorbed.15 This insight helped guide
strategies for more efficient drug delivery by inhibiting the
molecular pump.
Borst next turned to pseudoxanthoma elasticum, an inborn error
of calcification due to the absence of ABCC6 in the liver.16 This
genetic defect caused calcium to accumulate in the eyes, skin, and
blood vessels. Borst found that without functional ABCC6, there is a
lack of plasma pyrophosphate, which binds to calcium and prevents
calcification. This work guided development of new strategies for
overcoming drug resistance and improving therapeutic outcomes.
“If you want to discover new biology, it means that you
cannot only do the obvious experiments. You must sometimes
be a little more ingenious than the next person. So that means
that you sometimes tackle projects that nobody has much
faith in, and those projects often lead to real discoveries,”
Borst said.
Mentoring and advocating
Borst was deeply invested in training and mentoring numer-
ous students, many of whom have gone onto illustrious scientific
careers. “That’s the nice aspect of working within science. It’s a
combination of new biology and teaching biology. And I did a
lot of teaching in my life,” said Borst. “It’s a joint effort to, on one
hand, discover new things, and on the other hand, educate people
to have useful careers in later life.”
One of his previous graduate students, Titia de Lange, who
is now a renowned cell biologist at the Rockefeller University,
studied the genetic underpinnings of variant surface glyco-
proteins in trypanosomes under Borst’s guidance. During her
time in Borst’s lab, she noted that he was exceptionally good
at dissecting experiments and didn’t hold back from voicing
WINTER 2 02 3 | T H E S C IE N T IST 51
 PROFILE
his concerns. His deep commitment to science shone through
his actions.
“He is supremely adept at helping people achieve their best.
He had endless patience to sit with people and go through their
notebooks and look at the details of their experiments so he
could help figure out what was going wrong. He never gave up
on his trainees even though he was running the Dutch Cancer
Center—a huge job,” said de Lange.
He is supremely adept at helping people
achieve their best. He had endless patience
to sit with people and go through their note-
books and look at the details of their experi-
ments so he could help figure out what was
going wrong. He never gave up on his train-
ees even though he was running the Dutch
Cancer Center—a huge job.”
—Titia de Lange, The Rockefeller University
Aside from teaching, Borst served as the director of research
at the Netherlands Cancer Institute. At the time, the institu-
tion suffered from grant cuts, low productivity, and even lower
morale. He stepped in to address the problems with some sug-
gested changes. His vision to improve the institute faced stiff
opposition. For instance, Borst wanted to eliminate tenure for
graduate students because he thought that it locked people into
positions that were unsustainable. It was an unpopular stance,
but Borst insisted.
This conflict took Borst to court where he appealed to the
judges and convinced them that the institute could not func-
tion if graduate students worked in permanent jobs. Taking
advantage of this momentum, Borst also began to implement
other policies. To protect students, he established thesis com-
mittees to ensure the timely progression of students’ proj-
ects. He also implemented more stringent internal reviews
of grant applications before they were submitted to dramati-
cally improve funding success.
Borst played a prominent role in engaging with the gov-
ernment on scientific matters. Funding for basic research
paled in comparison to translational research. Borst appealed
for more funding for fundamental basic research as a neces-
sity to further exploit new knowledge that can be turned into
practical applications.
In addition, Borst firmly supported the need for science com-
munication. He frequently appeared on TV and radio and wrote
thought provoking columns in the Nieuwe Rotterdamsche Cou-
rant. “Effective communication is an important function of sci-
entists,” Borst said.
52 T H E SC I EN T I ST | the-scientist.com
“It is important to stress the power of science to solve prob-
lems,” Borst said. “Our society is so completely dependent on sci-
ence and technology that springs from that. If we don’t convince
people that the scientific method is the way to get at the truth,
then we won’t survive as humanity.”
In his five decades of scientific work, Borst contributed to
seminal discoveries in diverse topics, including mitochondria,
trypanosomes, and molecular pumps involved in multidrug
resistance. Borst’s deep passion for science extended beyond
his scientific research; he mentored numerous notable scien-
tists and played an active role in advocating for basic research
and scientific integrity. For his exceptional career of scientific
discovery, Borst received the 2023 Lasker~Koshland Award
for Special Achievement in Medical Science earlier this year. J
References
1. Borst P. The malate-aspartate shuttle (Borst cycle): how it started and
developed into a major metabolic pathway. IUBMB Life. 2020;72: 2241-2590
2. Borst P, Weissmann C. Replication of viral RNA, 8. Studies on the enzymatic
mechanism of replication of MS2 RNA. Proc Natl Acad Sci. 1965;54:982-987.
3. Van Bruggen EF, et al. Circular mitochondrial DNA. Biochim Biophys Acta.
1966;119(2):437-439.
4. Borst P, Ruttenberg GJ. Renaturation of mitochondrial DNA. Biochim Biophys
Acta. 1966;114:645-647.
5. Opperdoes FR, Borst P. Localization of nine glycolytic enzymes in a
microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett.
1977;80:360-364.
6. Hoeijmakers JH, et al. Novel expression-linked copies of the genes for variant
surface antigens in trypanosomes. Nature. 1980;284:78-80.
7. Bernards A, et al. Activation of trypanosome surface glycoprotein genes
involves a duplication-transposition leading to an altered 3’ end. Cell.
1981;27:497-505.
8. Van der Ploeg LH, et al. RNA splicing is required to make the messenger
RNA for a variant surface antigen in trypanosomes. Nucleic Acids Research.
1982;10:3591-3604.
9. Van der Ploeg LH, et al. Structure of the growing telomeres of Trypanosomes.
Cell. 1984;36:459-468.
10. Gommers-Ampt JH, et al. beta-D-glucosyl-hydroxymethyluracil: a novel
modified base present in the DNA of the parasitic protozoan T. brucei. Cell.
1993;75:1129-1136.
11. van Luenen H, et al. Glucosylated hydroxymethyluracil (DNA
base J) prevents transcriptional read-through in Leishmania. Cell.
2012;150(5):909-921.
12. Smit JJ, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene
leads to a complete absence of phospholipid from bile and to liver disease. Cell.
1993;75(3):451-462.
13. Deleuze JF, et al. Defect of multidrug-resistance 3 gene expression in
a subtype of progressive familial intrahepatic cholestasis. Hepatology.
1996;(4):904-908.
14. Smith AJ, et al. MDR3 P-glycoprotein, a phosphatidylcholine translocase,
transports several cytotoxic drugs and directly interacts with drugs as judged by
interference with nucleotide trapping. J Biol Chem. 2000;275(31):23530-23539.
15. Schinkel AH, et al. Normal viability and altered pharmacokinetics in mice
lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci.
1997;94(8):4028-4033.
16. Jansen RS, et al. ABCC6-mediated ATP secretion by the liver is the
main source of the mineralization inhibitor inorganic pyrophosphate
in the systemic circulation-brief report. Arterioscler Thromb Vasc Biol.
2014;34(9):1985-1989.
 PROFILE

Making Standards Exceptional

Samantha Maragh has taken on the difficult challenge of standardizing assays, data
norms, and terminology in the ever evolving genome editing field.

BY MEENAKSHI PRABHUNE, PhD

W
hen Samantha Maragh, who now leads the Genome
Editing Program at the National Institute for Stan-
dards and Technology (NIST), was in elementary
school, she loved watching the Forensic Files documentary
series on television with her father. Watching scientists, espe-
cially women, perform remarkable DNA analysis experiments
to solve criminal cases was her first exposure to the profession.
Maragh was inspired; the show sparked an early aspiration to
become a scientist as an adult. “It’s a TV show, but it was really
impactful to me,” Maragh recalled.
Maragh grew up in Baltimore, Maryland, as a first-genera-
tion American to immigrant parents. Her parents, having moved
from Jamaica to ensure opportunities for their children, priori-
tized her education throughout her school years. Maragh main-
tained a healthy interest in biology all those years, but when
she took her first genetics course in her undergraduate stud-
ies at Loyola University, she knew instantly that she had found
her calling. “As, Gs, Cs, and Ts and combining things together
doesn’t make any logical sense, but it completely resonated with
me, like I had found my science home,” she reminisced.
Despite her desire to keep learning, Maragh did not pursue
a master’s degree right away following graduation. Instead, she
planned to first secure a job and then ask her employers to fund
her continued education. Even though her friends were skepti-
cal, Maragh remained hopeful. As luck would have it, Maragh
secured a position as a technician at the NIST in May 2006. This Samantha Maragh leads the Genome Editing Program at NIST.
job shaped the course of her career path.

A tryst with NIST Maragh first worked on a collaborative project between

NIST is a nonregulatory federal agency under the US Department of
Commerce. Set up in 1901, the organization sets standards for mea-
surements for just about everything from atomic clocks to electrical
outlets. While the NIST organization integrated physics, chemistry,
and engineering into its working groups early on, the institution for-
mulated biology specific divisions relatively recently in the 1990s.
Maragh snagged a role at the Biochemical Sciences Division
but knew nothing about NIST when she applied. However, she
soon realized the value of the work done at the institute.
One of NIST’s projects involved standardizing the short tan-
SAMANTHA MARAGH
NCI and NIST to study cancer biomarkers for early detection.
Researchers reported new cancer biomarkers frequently, and yet
the subsequent experiments with these markers weren’t pan-
ning out for some reason. Maragh took up the task of developing
positive controls to ensure consistency and efficiency of assays,
specifically sequencing-based ones for identifying mitochon-
drial DNA mutations, used across different research groups.2
“She was just learning and growing at such a rapid pace and
had become a real thought leader,” recalled Laurie Locascio, who
led Maragh’s division and oversaw the NCI collaboration projects

dem repeat (STR) DNA analysis method used in forensics for
reliably identifying an individual.1 Maragh recalled the surreal
feeling when she realized that her workplace was integrally con-
nected to the forensic science that inspired her years ago—a rare
full circle moment.
at the time. Locascio is currently the director of NIST and the
undersecretary of Commerce for Standards and Technology. “It
was clear that she would be tremendously successful,” she added.
Impressed by Maragh’s work excellence, her bosses at NIST
supported her in taking night courses for continued education,

WINTER 2 02 3 | T H E S C IE N T IST 53
 PROFILE
and Maragh obtained her master’s degree in biotechnology from
John Hopkins University in May 2008. However, Locascio and
a few other NIST leaders had even bigger plans for her.
Locascio felt that an advanced degree and exposure to life out-
side of NIST would enhance Maragh’s career options. Locascio’s
boss back then, Willie May, agreed. May tapped her on the shoul-
der one day and said that she should pursue a PhD degree. Maragh
could choose any competency for her graduate studies as long as
she could apply that knowledge at NIST once she graduated.
Three months after completing her master’s degree, Maragh
enrolled in the human genetics PhD program at John Hopkins
University, manifesting the master plan that she had conjured up
as a student.
Crispier than CRISPR
During her graduate program, Maragh studied gene function in
early cardiac development in zebrafish. She routinely knocked down
protein expression using morpholinos, which are oligonucleotides
that bind to mRNA and obstruct translation. Since knockdowns
don’t guarantee complete protein inhibition, Maragh wanted to vali-
date her results by suppressing protein expression at a genetic level.
She first attempted to use the zinc finger nuclease (ZFN) sys-
tem for genome editing but found it challenging. She then tried
to knock out her desired gene using transcription activator-like
effector nucleases (TALEN), a new genome editing tool at the
time with growing popularity.3 Using TALEN, Maragh repli-
cated the phenotype she had observed with RNA knockdown.
Around that time, she heard about a new study where
researchers had leveraged clustered regularly interspaced pal-
indromic repeats (CRISPR) and an associated nuclease, which
function as a natural immunity mechanism in bacteria, to create
a programmable machinery to edit cells.4 Although Maragh did
not get the chance to test the CRISPR system in the lab, she real-
ized the power the flexible technology offered right away, espe-
cially having worked with the relatively more complicated ZFN
and TALEN tools. As the list of CRISPR applications grew with
researchers globally adopting the technique, she also saw the
risk for inconsistencies and raised questions that not many were
thinking about at the time.
“What in the world are we doing to the genome? And how
do we know what we are doing?” she recalled wondering. “My
NIST brain went ‘controls, variability.’”
When Maragh returned to NIST, she had the opportunity
to propose a promising topic area that wasn’t on the organiza-
tion’s radar at the time: CRISPR. “This is a totally new world
and there’s a place for NIST here,” she thought.
When she pitched her idea at her division’s proposal meet-
ing to solicit peer feedback, a colleague asked, “Is there going
to be something crispier than CRISPR one day?” That got her
thinking. CRISPR was a promising tool, but ZFN and TALEN
remained important players in the field. And given the rapid sci-
entific advances in CRISPR-related technologies, it seemed likely
that newer tools were on the horizon.
5 4 T H E SC I EN T I ST | the-scientist.com
When I heard about NIST and their mission
and Samantha's interest in having NIST take
a role in doing this, I thought, yes, that would
be fantastic.
—Keith Joung, Massachusetts General Hospital
Maragh broadened her scope to genome editing, success-
fully convinced her colleagues about the promising outlook of
this area, and eventually started the Genome Editing Program
at NIST in 2016. The program aims to apply the NIST lens of
standards to genome editing research to ensure that scientists
use consistent methodology, controls, data formats, and termi-
nology to minimize variability in experiments.
The first task for Maragh’s program team was to engage
with the genome engineering community and demonstrate that
NIST’s goals aligned with their needs. Around this time, Maragh
serendipitously came across a short commentary written by an
academic about the need for standards in CRISPR research.5
She knew right away that she had found a potential ally: Keith
Joung, a genome editing pioneer and group leader at Massachu-
setts General Hospital.
A consortium is born
Joung has been strategizing genome and epigenome editing technol-
ogies that could improve human health for almost 20 years. With the
rapid growth in the sector at the time, Joung worried about exper-
imental technique variability between groups. He saw a need for
standardizing definitions and practices to establish a baseline for
the researchers working in this emerging area. “When I heard about
NIST and their mission and Samantha’s interest in having NIST take
a role in doing this, I thought, yes, that would be fantastic,” he said.
With Joung’s support, Maragh conducted her first workshop
at the American Society of Gene and Cell Therapy Conference in
2016. Representatives from across the biotechnology sector, aca-
demia, pharmaceutical companies, and nonprofit organizations
attended. Maragh sourced feedback regarding their challenges
and interrogated where they
needed data validation tools.
“I heard a lot of positivity
there. One organization was
like, ‘You know, I can see their
building, but I can’t go talk to
them because of noncompete
sort of things, and I need a
mechanism that would allow
us to be under the same
umbrella,’” Maragh recalled.
Maragh thought that a
Maragh set up the genome editing
program at NIST after completing
consortium would be the
her graduate studies. perfect solution. However, on
NIST
soliciting further feedback,
she realized that the genome editing community needed more than
just a forum that convened to exchange ideas. They sought NIST’s
support in making samples and executing experiments.
When Maragh realized that this meant that NIST would
almost function as their research arm, she worried about how
she would find resources for these unconventional requirements.
Determined to find a creative solution, Maragh conceptualized
a new cost-sharing consortium model where every member contrib-
uted in some way. “If you are a sequencing company, maybe you
can sequence. If you are a reagent maker, maybe you can provide
reagents. If you are an engineering company, maybe you can support
with some cell engineering, and if you are big pharma, maybe you
just give money,” she explained. “It was very different for NIST. There
was no such model; it took me a while. I had to create the model.”
After months of grueling paperwork, Maragh officially launched
the Genome Editing Consortium in October 2018 with 20 members
from diverse institutions. The consortium comprised three working
groups. The “specificity measurements” group worked on the repro-
ducibility of on-target and off-target assays and related controls. The
“data and metadata” group covered bioinformatics related topics such
as evaluating different tools or generating high quality data sets that
researchers could use as positive controls. Lastly, the “lexicon” team
standardized the genome editing vocabulary. Members could join one
or more groups based on their needs, and each group met monthly.
Bringing together a disparate group of people who have differ-
ent interests in working together is not an easy task, commended
Joung. “I think she’s done a great job of that.”
Locascio seconds that opinion. “I really give great credit to
her for thinking about this and what was a very nascent field
and building this consortium around what could be the diffi-
culties in getting this implemented in the real world,” she said.
The consortium has made big strides in several projects
over the past few years. Maragh is particularly proud of com-
pleting the first version of a genome editing vocabulary stan-
dard that includes definitions of key terms used in the field.
The team worked hard on curating the relevant terms, draft-
ing the concise definitions, getting experts to review them, and
finally seeking global feedback. After a regimented and rigor-
ous review process, Maragh was thrilled when the document
was finally approved by the International Standards Organi-
zation and published on their site.
For her success in building the NIST Genome Editing Consor-
tium as a public-private partnership, Maragh received the George
A. Uriano Award in 2021.
Staying on target and under control
Today, the genome editing consortium has grown to more than
40 members, and all three groups are still active. Joung enjoys
watching Maragh in action bringing members with diverse
interests to work together. “The advances come so quickly that
to some degree, you have to be nimble and adjust or expand
your expectations as far as the field’s,” he said. “I’m grateful that
somebody like her is willing to undertake this type of effort.”
For instance, within just a few years after CRISPR being
adopted in labs worldwide, the genome editing community wel-
comed new tools such as base editors and prime editors. So, the
standardized lexicon document lacks the definitions for these
technologies, which didn’t exist at the time of its creation, but
are now crucial to the field. Maragh hopes that her teams will
add suggested definitions for some of the missing terms to the
glossary at some point.
In fact, there is a long wishlist of goals she would like to accom-
plish in the near future. In one project, her team intends to generate
some physical samples of engineered cells bearing arrays of edits for
consortium members to use as positive controls for complex edits.
Maragh spoke of another interesting consortium project that is
currently underway. This Genome in a Bottle (GIAB) project is a
blind inter-lab study to assess the accuracy of capabilities that con-
sortium members are currently using for detecting DNA variants.
The NIST team sent consortium researchers a mixture of cells or
DNA with varying sizes and frequencies of mutations at different
loci. The researchers will use the technology of their choice and
expertise, such as sequencing, genome wide DNA imaging, and
fragment analysis, and report back the variant size and frequency
data. On comparing data from different research groups, Maragh
hopes that the NIST team can zero in on the most accurate capa-
bilities that will serve as the gold standard for detecting DNA vari-
ants moving forward.
“The goal of setting standards for how things are measured
in our field remains a very, very important one. And so, I would
like to see them be a bit more vocal and proactive in trying to put
these standards out there and trying to get people in the field to
follow them,” Joung suggested.
In addition to leading the Genome Editing Program, Maragh
now comanages NIST’s Cancer Biomarker and Genomic Sci-
ence Group, which includes programs on genome editing, can-
cer biomarkers, flow cytometry, and human genome sequencing.
Although she does not have a master plan for the future anymore,
she wants to continue working in the regenerative medicine and
precision medicine application areas.
“She is clearly a scientific leader, and now she leads an exter-
nal facing consortium, but I definitely see her growing into larger
roles and moving up the organization,” said Locascio. “She just
has very unique characteristics that make her a natural leader.” J
References
1. Ruitberg CM, Reeder DJ, Butler JM. STRBase: a short tandem repeat DNA
database for the human identity testing community. Nucleic Acids Res.
2001;29(1):320-322.
2. Jakupciak J, et al. Analysis of potential cancer biomarkers in mitochondrial
DNA. Curr Opin Mol Ther. 2006;8(4):345-354. Accessed August 1, 2023.
3. Joung JK, Sander JD. TALENs: a widely applicable technology for targeted
genome editing. Nat Rev Mol Cell Biol. 2013;14(1):49-55.
4. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in
adaptive bacterial immunity. Science. 2012;337(6096):816-821.
5. Joung J. Standards needed for gene-editing errors. Nature. 2015;523:158
WINTER 2 02 3 | T H E S C IE N T IST 5 5
 METHODS
Whenever, Wherever: Taking DNA
Amplification Outside the Lab
Recombinase polymerase amplification lets researchers rapidly replicate DNA in the clinic,
in the field, or even in the International Space Station.
BY HANNAH THOMASY, PhD
C
etus biochemist Kary Mullis’ invention of polymerase
chain reaction (PCR) in 1985 revolutionized genetics and
won him a share of the 1993 Nobel Prize in Chemistry.1
PCR was instrumental in the sequencing of the human genome,
and today, it is used for everything from diagnosing genetic disor-
ders to studying the evolutionary relationships between species.2,3
Despite its utility, PCR’s main limitation is that it requires
precise cycles of heating and cooling to amplify DNA. The ther-
mal cyclers that perform this operation are clunky, relatively
expensive, and consume a lot of power, making them unsuitable
for use in the field or in low-resource settings.
In 2006, researchers Olaf Piepenburg, Colin Williams, and
Niall Armes of ASM Scientific, which later became TwistDx,
along with Derek Stemple, at the time a molecular biologist at
the Wellcome Trust Sanger Institute and currently the chief sci-
ence officer at Camena Bioscience, developed an alternative. They
invented recombinase polymerase amplification (RPA), an iso-
thermal amplification technique that functions at relatively low
temperatures.4 “Just the warmth of your hand is enough to drive
the reaction,” said Stemple.
Since its invention, researchers have demonstrated a myriad
of applications for this technology, including rapid identifica-
tion of plant, animal, and human pathogens, including SARS-
CoV-2 , detecting antimicrobial resistance genes, and water qual-
ity monitoring.5–10
The beginnings of RPA
The researchers didn’t originally set out to develop a new isother-
mal amplification method, said Piepenburg. Instead, they were
trying to develop a new technique for single molecule sequencing.
During this project, they became interested in ways to sequence
specific sites within the genome, rather than random pieces.
“Niall’s idea was to use recombinase proteins like RecA to
target DNA primers to specific sites,” said Piepenburg. “This
idea evolved into the notion that if you have two primers that are
interacting with recombinase proteins and they move in opposite
directions along a double-stranded piece of DNA, wouldn’t you
start to get an amplification reaction?”
RecA is a bacterial protein that is crucial for repairing dou-
ble-stranded breaks in DNA via the process of homologous
recombination. RecA binds to single-stranded DNA, searches
along a separate piece of double stranded DNA for the cognate
5 6 T H E SC I ENTI ST | the-scientist.com
sequence, and then promotes DNA strand invasion.11 After test-
ing various proteins in the RecA family, the researchers ulti-
mately settled on the phage protein uvsX recombinase, which
performs a similar function. In RPA, this recombinase protein
binds to primers designed by scientists, then scans the DNA,
and inserts the primers into complementary sites in the DNA.
Then single-stranded binding proteins called gp32 bind to the
displaced DNA strand to stabilize it. The recombinase disas-
sembles, allowing a DNA polymerase to bind to the 3’ end of
the primer, and DNA synthesis begins.
“We tested a lot of different polymerases,” said Stemple.
“The polymerases that worked the best were the ones that
were able to displace the second strand, that don’t need sin-
gle-stranded DNA to work from, like Taq polymerase.” The
MODIFIED FROM © ISTOCK.COM, BEZVERSHENKO, FEDORA BRADAS, VITALII DUMMA, INVINCIBLE_BULLDOG
researchers eventually found a polymerase from Bacillus sub-
tilis that was especially effective. “It’s like a cow catcher at the
front of the train, separating double stranded DNA at the front
of the polymerase,” said Stemple.
This technique enables rapid amplification of the target DNA:
the process takes just 20 to 40 minutes.12 Once the target DNA has
been amplified, researchers can use a variety of strategies to detect
the DNA, including lateral flow and fluorescence-based tests.
The researchers also demonstrated the real-world utility of
this technique. “We really wanted to show that we could detect
disease-causing bacteria in very small numbers,” said Stemple.
Indeed, by combining RPA with a fluorescent probe detection
system, they detected just ten copies of DNA from methicillin-
resistant Staphylococcus aureus (MRSA), a pathogen responsible
for an estimated 100,000 deaths per year.13
“After we published the paper, we got an email from Kary
Mullis congratulating us,” said Stemple. “That was pretty cool.”
Detecting DNA from the cervix to outer space
“A question that we faced early on when presenting at scientific
conferences or talking to potential collaborators was, ‘Why not just
use PCR?’” said Piepenburg. “PCR is excellent at what it does, but
it is limited by the fact that you need a heavy instrument that takes
a lot of energy to run. So, it’s very hard to see PCR ever reaching a
stage where you can make a completely disposable diagnostic. And
especially for rapid diagnostics, that’s really the holy grail.”
With RPA in their toolkit, researchers around the world can
now develop rapid tests for various plant, animal, and human
RECOMBINASE POLYMERASE AMPLIFICATION IN ACTION
A rapid isothermal amplification technique enables pathogen identification and antibiotic resistance detection in low-resource settings.

Recombinase polymerase amplification (RPA) is a technique for rapidly copying segments of DNA. Unlike polymerase chain reaction (PCR),
it does not require thermal cycling, so less equipment is needed.

HOW RPA WORKS RPA APPLICATIONS

Researchers require three defining elements for RPA: uvsX recombinase proteins, single- Because RPA is rapid, portable, and runs at low
stranded binding proteins, and strand-displacing DNA polymerases. They also need primers temperatures it has diverse applications.
and nucleotides to amplify the target DNA.

Recombinase Optimal temperature

proteins Recombinase- 37-42° C
primer complexes

Primers

Point-of-care testing for genetic

and infectious diseases

DNA
Strand-displacing
DNA polymerase

Q
1 First, the primers bind to the Q4 The recombinase disassembles and
uvsX recombinase proteins to form the strand-displacing DNA polymerase
recombinase-primer complexes. binds to the 3’ end of the primer.

Water quality testing

Q2 Then the recombinase inserts the primers Q

5 The polymerase elongates the primer.
into complementary sites in the DNA.
Antibiotic resistance detection

Single-stranded
binding proteins

Q
3 Single-stranded binding proteins bind to Q
6 The target DNA strands are duplicated.
the displaced DNA strand and stabilize it. Agricultural pathogen monitoring
 METHODS
pathogens that can be used in places far afield from traditional
laboratory settings, with important implications for both agricul-
ture and human health.7,14,15
One such application is human papillomavirus (HPV) test-
ing, which has long been difficult in low-resource settings. “There
is a huge need for point-of-care technologies that can be used
anywhere in the world; remote locations and developing coun-
tries are really in need of tests that can accurately detect HPV in
women,” said Sylvia Daunert, a biochemist and molecular biolo-
gist at the University of Miami.
Just the warmth of your hand is enough to
drive the reaction.
—Derek Stemple, Camena Bioscience
The test also needs to be fast. In these settings, said Daunert,
“if women leave the community health clinic without being
treated, the likelihood that they will come back is very low.”
“What makes RPA really good for this application is that it
works at just one temperature, which is relatively low compared
to other isothermal methods, some of which need to be kept
at 65 degrees Celsius or require initial heating,” said Daunert.
Daunert and her team designed consensus primers that could
amplify any of the 14 high-risk HPV genotypes. They lyophilized the
reagents and combined them with primers into one tube, reducing
the number of steps and eliminating the need for precise pipetting.
“We wanted anyone to be able to operate this test,” said Daunert.
Their test detected high-risk HPV with a sensitivity of 96 per-
cent and a specificity of 83 percent.7 Daunert’s next step will be
testing the diagnostic on the ground in low-resource settings in
regions like central Africa.
Some researchers deploy RPA even farther afield. Gur Pines,
who studies molecular diagnostics and detection at the Agri-
cultural Research Organization–Volcani Institute, was working
on a rapid molecular detection strategy for the peach fruit fly,
an important agricultural pest, when he got the opportunity to
send an experiment to space as part of Israel’s Rakia Mission.
Pines studied a detection strategy that combined RPA and
CRISPR technology. RPA amplifies the DNA, and when CRISPR-
Cas12a binds to the target DNA, the Cas12a begins to indiscrimi-
nately chop up single-stranded DNA. (This nonspecific cutting of
single-stranded DNA is unique to Cas12a and is not shared by the
more well-known Cas9 protein.) Researchers use probes with a
fluorophore and a quencher attached by single-stranded DNA, so
when the target DNA is detected, Cas12a cleaves the probe, free-
ing the fluorophore and producing a fluorescent signal.
This technique, dubbed DETECTR, was originally developed by
Jennifer Doudna, a biochemist at the University of California, Berke-
ley and the codeveloper of CRISPR-Cas9 genome editing, and her
team in 2018, but it had not previously been tested in microgravity.16
“We wanted to test whether this technology worked in micro-
gravity because it is such an awesome technology,” said Pines.
5 8 T H E SC I ENTI ST | the-scientist.com
“It has so many potential applications and it could be ideal for
long space missions. They don’t need to use fancy machines or
send the samples back to Earth. They can have results almost
in real time.”
However, said Pines, “We don’t fully understand the effects of
microgravity on biological reactions.” So, researchers designed a
proof-of-concept study to test whether this technique could iden-
tify synthetic genomic sequences from three different species: a
bacterium, a fungus, and an insect.
The researchers found that the test was still extremely sen-
sitive in microgravity: it identified the target DNA in attomolar
concentrations.17 In the future, researchers could use this tech-
nology to design kits to diagnose diseases in astronauts, study
how the microbiome of the station and its inhabitants changes
over time, or monitor the health of space farming operations
on long missions. J
References
1. The Nobel Prize in Chemistry 1993. NobelPrize.org.
2. Polymerase Chain Reaction (PCR) Fact Sheet. Genome.gov.
3. Suyama Y et al. Complementary combination of multiplex high-throughput DNA
sequencing for molecular phylogeny. Ecological Research. 2022;37(1):171-181.
4. Piepenburg O et al. DNA Detection Using Recombination Proteins. PLoS Biol.
2006;4(7):e204.
5. Silva G et al. Rapid and specific detection of Yam mosaic virus by reverse-
transcription recombinase polymerase amplification. J Virol Methods.
2015;222:138-144.
6. Zhao G et al. Development of a recombinase polymerase amplification
combined with a lateral flow dipstick assay for rapid detection of the
Mycoplasma bovis. BMC Veterinary Research. 2018;14(1):412.
7. Seely S et al. Point-of-Care Molecular Test for the Detection of 14 High-
Risk Genotypes of Human Papillomavirus in a Single Tube. Anal Chem.
2023;95(36):13488-13496.
8. Nelson MM et al. Rapid molecular detection of macrolide resistance. BMC
Infectious Diseases. 2019;19(1):144.
9. Luo N et al. Establishment of methods for rapid detection of Prymnesium
parvum by recombinase polymerase amplification combined with a lateral
flow dipstick. Frontiers in Marine Science. 2022;9.
10. Liang L et al. Development of a multi recombinase polymerase amplification
assay for rapid identification of COVID 19, influenza A and B. J Med Virol.
Published online September 20, 2022:10.1002/jmv.28139.
11. Cox MM. Motoring along with the bacterial RecA protein. Nat Rev Mol Cell
Biol. 2007;8(2):127-138.
12. Lobato IM, O’Sullivan CK. Recombinase polymerase amplification: Basics,
applications and recent advances. Trends Analyt Chem. 2018;98:19-35.
13. Murray CJL et al. Global burden of bacterial antimicrobial resistance in 2019:
a systematic analysis. The Lancet. 2022;399(10325):629-655.
14. Strayer-Scherer A et al. Recombinase Polymerase Amplification Assay
for Field Detection of Tomato Bacterial Spot Pathogens. Phytopathology.
2019;109(4):690-700.
15. Conrad CC et al. A Sensitive and Accurate Recombinase Polymerase
Amplification Assay for Detection of the Primary Bacterial Pathogens Causing
Bovine Respiratory Disease. Frontiers in Veterinary Science. 2020;7.
16. Chen JS et al. CRISPR-Cas12a target binding unleashes indiscriminate single-
stranded DNase activity. Science. 2018;360(6387):436-439.
17. Alon DM et al. CRISPR-based genetic diagnostics in microgravity. Biosens
Bioelectron. 2023;237:115479.
 METHODS

Finding the One in a Million

Phage display revolutionized peptide screening methods and unlocked opportunities in
protein discovery and development.

BY SHELBY BRADFORD, PhD

W
hen recombinant protein expression was introduced
in 1977, screening clones was a laborious and time-
consuming process.1-3 While on a sabbatical at Duke
University, George Smith, a biochemist at University of Missouri,
reasoned that he could improve this process by leveraging a coat
protein, called pIII, from the filamentous bacteriophage that he
studied. He tested his initial design with two Duke University bio-
chemists, Robert Webster and Paul Modrich. Their collaboration
birthed phage display, a combinatorial marvel that Smith referred
to in his Nobel lecture as “simple evolution in a Petri dish.” It com-
pletely changed the field of recombinant protein biology.

Use what you have

In the early and mid-1980s, scientists used phages to express
recombinant proteins. The protein was retained inside the viri-
ons, so screening required growing viral plaques, creating a stamp
of these on a membrane, treating the membrane with antibody,
and then mapping antibody labels on the membrane back to the
exact viral plaques on the Petri plate.
Smith’s vision for improving recombinant protein analy-
sis was to express a foreign protein at one end of pIII and then
select for it using an antibody affixed to a surface. In this way,
scientists could retain only positive clones and immediately use
their selected phage to generate more phages. This made screen-
ing higher throughput, as researchers could screen thousands or
even millions of clones simultaneously.
It completely opened the door to making fully
human antibodies as therapeutics.
—John McCafferty,
Cambridge Institute of Therapeutic Immunology and Infectious Disease
Additionally, because the protein would be bound to the bac-
teriophage with the inserted DNA, the product and instructions
© ISTOCK.COM, MIRROR-IMAGES
Scientists use phage display to generate monoclonal antibodies.
mentous phage display.4 In subsequent years, Smith and his team
developed a peptide library in which five to six amino acid resi-
dues in a peptide sequence were randomized.5 In this work, they
demonstrated the value of phage display in selecting for high-
binding peptides through a series of affinity purification steps,
where bound phages were eluted, amplified in a bacterial host,
and then reintroduced to their target ligand to select for binders.
These libraries allowed for screening potentially billions of ran-
dom epitopes simultaneously.
While Smith and his colleagues’ peptide libraries demonstrated
the potential of phage display, the sequences were randomly gen-
erated. Scientists did not have control over the amino acid fre-
quencies, and because of redundancies in amino acids, could have
overrepresentation of some residues. To improve control over the

for it were obtained at the same time as a genotype-phenotype
linkage. “You can then sequence that particular phage clone, and
it will tell you the nucleotide sequence and therefore, the amino
acid sequence of the peptide displayed on its surface,” said Jamie
Scott, who joined Smith’s lab as a postdoctoral fellow in the late
1980s and is now a professor emeritus at Simon Fraser University.
When Smith returned to his lab, he explored this method
further, and in 1985, published his work on what he called fila-
amino acids used and circumvent the introduction of stop codons,
subsequent groups developed methods to synthesize codons indi-
vidually.6 These presynthesized codons can be mixed at specific
percentages to yield a library with the characteristics researchers
are most interested in, such as polarity and binding affinities.
“It’s not random,” said Anthony Kossiakoff, a protein engi-
neer at the University of Chicago who studies protein structure-
function relationships. “There’s a lot of planning and designing

WINTER 2023 | T H E S C IE N T IST 59

PHAGE DISPLAY ALLOWS RAPID SCREENING OF MILLIONS OF PEPTIDES
A viral protein expression method links proteins and their coding instructions, enabling easier target identification for downstream analysis.

Q
1 PHAGE DISPLAY LIBRARY GENERATION

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
Transformation

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
+ Coat
protein

Bacteria with Displayed protein

DNA library Plasmid library plasmid Helper phage Phage library

Q
2 AFFINITY PURIFICATION
////////////////////////////////

////////////////////////////////
////////

////////
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////

Wash Elute
////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
////////////////////////////////
////////////////////////////////

////////////////////////////////

////////////////////////////////
////////////////////////////////

////////////////////////////////

////////////////////////////////
////////////////////////////////
////////

////////
////////

////////
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

C
C
C
C
C
C
C
C

C
C
C
C
C
C
C
C
Eluted phage
Phage library Target binding Displayed
C
C protein Discard

////////////////////////////////

////////////////////////////////
////////////////////////////////

////////////////////////////////
Target ligand

Unbound phage

Q
3 PHAGE AMPLIFICATION
////////////////////////////////

////////////////////////////////

Affinity
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
purification Elute
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
+ +
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////
////////////////////////////////

////////////////////////////////

C
C
C
C
C
C
C
C
C
Eluted phage
Eluted phage Bacteria Helper phage

Q
4 CLONE ISOLATION

Purify phage
////////////////////////////////

////////////////////////////////

////////////////////////////////

////////////////////////////////

Sequence analysis
plasmid

+ Monoclonal antibody production

Protein analysis
Phage-infected bacteria

Q1 Scientists insert variable DNA sequences coding for their proteins of interest into plasmids that carry a phage coat protein gene, an antibiotic
resistance gene, and a packaging signal. Then they transform the plasmids into a bacterial vector and infect it with a helper phage that supplies the
other necessary viral proteins. This generates the phage library, where each virion expresses versions of the protein of interest on its coat protein and
carries its genetic sequence in its genome.

Q2 In the next step, researchers isolate the phages expressing surface proteins using a surface ligand binding assay. The final eluted phages may bear
a mix of different surface protein epitopes.

Q
3 In the amplification step, researchers propagate the eluted phages in a bacterial culture, and may run additional rounds of affinity purification.

Q
4 Researchers infect bacteria with the selected phages and culture them on plates containing antibiotics. In the final step, they pick resistant colo-
nies to isolate the plasmids, which are then sequenced and cloned into the desired protein production vectors for various applications.
 for how you can best utilize what you got.” Proficient structural
biologists consider more than the library.
“The biggest thing about any of these selections is the qual-
ity of the antigen,” Kossiakoff said. He explained that for phage
display to yield usable products, teams using the method need to
determine the stability of the antigen and ensure that it is in the
desired conformation.
Lastly, to select for high-affinity proteins, researchers must
develop adequate stringency conditions in their affinity puri-
fication steps. This may include decreasing the concentration
of ligand, introducing competing enzymes, screening against
ligands in other conformations, or blocking potential binding
sites of the target ligand during the maturation step.7
At the end, because of the genotype-phenotype linkage, a
researcher can extract the protein’s genetic sequence for a vari-
ety of downstream purposes.
Opening the Door
This linkage of the instructions with the binding product, coupled
with the ability to evaluate huge volumes of candidates, became
an attractive model for many research applications, but particu-
larly for antibody research and production.
While Smith’s method generated random peptide sequences
and tested them against known antibodies, other groups consid-
ered alternative approaches. “Wouldn’t it be really cool if we could
do that the other way around?” said John McCafferty, currently a
biologist at the Cambridge Institute of Therapeutic Immunology
and Infectious Disease and chief executive officer of Maxion Thera-
peutics. He and his postdoctoral mentor, Greg Winter, a molecular
biologist currently at the Medical Research Council Laboratory of
Molecular Biology, used phage display to produce antibodies against
known targets.
By cloning the variable regions of the heavy and light
INFOGRAPHIC: DESIGNED BY ERIN LEMIEUX; © ISTOCK.COM, LOVE EMPLOYEE
chains of antibodies from immunized blood donors together
on a single coding region, the two produced a fusion pro-
tein with all of the antigen binding properties of a full anti-
body, which they called single chain Fv (scFv). This scFv was
expressed by the bacteriophage and could be affinity selected
analogously to Smith’s peptides by using the immobilized
peptide as a binding target for the antibodies. From the
inception of this idea to the publication of the first success-
ful antibody took one year8—“A feat that I’ve tried to repeat
ever since without success,” McCafferty said.
Producing monoclonal antibodies was previously possible
using hybridoma methods.9 However, these methods could be
laborious and take months to immunize animals, isolate and cul-
ture B cells that were fused to immortalized cells, and then screen
them. With phage display, it was possible to isolate the genetic
material from B cells from immunized animals or humans and
immediately begin amplifying it in bacteriophages. Not only was
this faster, it also simplified the process of studying the genetic
sequence of these antibodies. Additionally, it alleviated the prob-
lems of hybridoma technology for producing animal antibod-
ies that had to be humanized. The binding sequences identified
through phage display could be isolated, reinserted into an exist-
ing human IgG backbone, and expressed in bacterial or mamma-
lian cells. “It completely opened the door to making fully human
antibodies as therapeutics,” McCafferty said.
This method led to the development of the world’s first anti-
body produced by phage display, the anti-TNF antibody adalim-
umab, more commonly known as Humira, which was produced
by McCafferty’s and Winter’s company Cambridge Antibody Tech-
nology with the help of Baden Aniline and Soda Factory (BASF).
Coupled with next-generation sequencing, phage display
allows for the rapid production and assessment of antibodies
Filamentous phages are used to express recombinant proteins in phage display.
 METHODS
It’s an extremely powerful technique to do all
kinds of things.
—Anthony Kossiakoff, University of Chicago
against an array of targets, including those that would otherwise
be impossible to produce in a human system. “Because in a syn-
thetic library, it has no basis for tolerance,” said Scott.
An extremely powerful technique
Phage display, like most technologies, has evolved. In the world of
antibody production, researchers explored display vehicles such
as nontraditional viruses and bacteria and also used eukaryotic
cells, such as yeast and mammalian cell lines.10,11
“In some ways, a phage display system is a bit of a black box,”
McCafferty said. “You’ve got your library; you’ve got your antigen;
you’ve been through a process; and out comes stuff at the other
end.” While one can easily find a slew of binding candidates, it’s
often more than can be easily or desirably assessed for detailed
binding affinity or cross-recognition with other potential targets.
While yeast and mammalian cell display methods do not offer
the scale of library potential that phages do, these approaches
are compatible with flow cytometry and cell-sorting applica-
tions, which allow researchers to explore far more of their pro-
tein-expressing clones and their biophysical properties.
Researchers also use alternative backbones for their peptides
beyond the traditional immunoglobulin scaffolds. These include
small proteins or even domains of proteins, such as fibronectin
binding domain, which can support the introduction of vari-
able peptide regions.12 These new scaffolds can be smaller than
immunoglobulin and overcome structural limitations, such as
the immunoglobin’s dual-chain design, a double cysteine bond,
and necessary post-translational modifications.13,14
Modified selection methods also expand the capacity to select
displayed proteins with desired characteristics. This includes in
vivo selection, where candidates are delivered to a model animal
and their organ distribution is assessed, and whole cell culture,
where the library can be screened against multiple cell types to
determine receptor recognition.15-17
Beyond antibodies, researchers use phage display to explore
and manipulate protein interactions, either to study protein
functions or to apply toward novel drug discovery.18,19 “You can
ask questions that are very outside the box about the energetics
of interfaces and understand, as evolution has put these things
together, what was really important, and why it’s important and
conserved,” Kossiakoff said.
Phage display was used to identify rare or even unnatural
peptides for cancer therapeutics and B cell epitopes in disease
and to screen against allergens to characterize them or chemi-
cal compounds to study their mechanism of action.20-23 It’s also
being explored for identification and production of antivenom
antibodies, some of which are cross protective against venom
6 2 T H E SC I ENTI ST | the-scientist.com
from multiple snake genera.24,25 “It’s an extremely powerful tech-
nique to do all kinds of things,” Kossiakoff said. J
References
1. Itakura K, et al. Expression in Escherichia coli of a chemically synthesized
gene for the hormone somatostatin. Science. 1977;198(4321):1056-1603
2. Mierendorf RC, et al. Gene isolation by screening gt11 libraries with
antibodies. Methods Enzymol. 1987; 152: 458-469
3. Young RA & Davis RW. Efficient isolation of genes by using antibody probes.
Proc Natl Acad Sci. 1983;80(5):1194-1198
4. Smith GP. Filamentous fusion phage: novel expression vectors that display
cloned antigens on the virion surface. Science. 1985;228(4705):1315-1317
5. Scott JK & Smith GP. Searching for peptide ligands with an epitope library.
Science. 1990;249(4967):386-390
6. Virnekas B, et al. Trinucleotide phosphoramitides: ideal reagents for the
synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids
Res. 1994;22(25):5600-5607
7. Pande J, et al. Phage display: concept, innovations, applications, and future.
Biotechnol Adv. 2010;28(6):849-858
8. McCafferty J, et al. Phage antibodies: filamentous phage displaying antibody
variable domains. Nature. 1990;348:552-554
9. Mitra S & Tomar PC. Hybridoma technology; advancements, clinical
significance, and future aspects. J Genet Eng Biotechnol. 2021;19(159)
10. McMahon C, et al. Yeast surface display platform for rapid discovery of
conformationally selective nanbodies. Nat Struct Mol Biol. 2018;25:289-296
11. Robertson N, et al. Development of a novel mammalian display system
for selection of antibodies against membrane proteins. J Biol Chem.
2020;295(52):18436-18448
12. Gilbreth RN & Koide S. Structural insights for engineering binding proteins
based on non-antibody scaffolds. Curr Opin Struct Biol. 2012;22(4):413-420
13. Binz HK & Plückthun. Engineered proteins as specific binding reagents. Curr
Opin Struct Biol. 2005;16(4):459-469
14. Skerra A. Imitating the humoral immune response. Curr Opin Chem Biol.
2003;7(6):683-693
15. Kolonin MG, et al. Reversal of obesity by targeted ablation of adipose tissue.
Nat Med. 2004;10:625-632
16. Pasqualini R & Ruoslahti E. Organ targeting in vivo using phage display
peptide libraries. Nature. 1996;380:364-366
17. Barry MA, et al. Toward cell-targeting gen therapy vectors:selection of cell-
binding peptides from random peptide-presenting phage libraries. Nat Med.
1996;2:299-305
18. Sidhu SS, et al. Exploring protein-protein interactions with phage display.
Chembiochem. 2003;4(1):14-25
19. Krumpe LRH & Mori T. Potential of phage-display peptide library technology
to identify functional targeting proteins. Expert Opin Drug Discov.
2007;2(4):525-537
20. Brown KC. Peptidic Tumor Targeting Agents: The Road from Phage
Display Peptide Selections to Clinical Applications. Curr Pharm Des.
2010;16(9):1040-1054
21. Dybwad A, et al. Identification of new B cell epitopes in the sera of rheumatoid
arthritis patients using a random nanopeptide phage library. Eur J Immunol.
1993;23(12)3189-3193
22. Rhyner C, et al. Cloning allergens via phage display. Methods.
2004;32(3)212-218
23. Van Dorst B, et al. cDNA phage display as a novel tool to screen for cellular
targets of chemical compounds. Toxicol in Vitro. 2010;24(5):1435-1440
24. Ahmadi S, et al. An in vitro methodology for discovering broadly-neutralizing
monoclonal antibodies. Sci Rep. 2020;10:10765
25. Ledsgaard L, et al. Discovery and optimization of a broadly-neutralizing
human monoclonal antibody against long-chain-_-neurotoxins from snakes.
Nat Commun. 2023;14:682
Hello World!
The New TS Digest is Here
Our new interactive TS Digest is the literary
equivalent of a fine dining tasting menu. We have
created bite-sized content pieces in diverse formats,
crafted with the convenience of the reader in mind.
You can now quickly browse a brief news story, play
a video clip, solve a crossword puzzle, or peruse an
infographic—all during your short coffee break.

EXPLORE NOW!

BROUGHT TO YOU BY
2024
PROGRAMS
ANTIBODY DISCOVERY
& ENGINEERING JANUARY 16-19, 2024 | SAN DIEGO, CA HILTON SAN DIEGO BAYFRONT

%,63(&,),&$17,%2'<
'(9(/230(17 T HE PROTEIN SC IENC E AND PRODUCTION WE E K
CHARACTERIZATION
& AGGREGATION IN
%,23+$50$&(87,&$/6

VECTOR DESIGN REGISTER

& DELIVERY
NOW FOR
+,*+(5Ǩ7+528*+387
%,2352'8&7,21
ADVANCE
SAVINGS
TRAINING SEMINARS

Organized by

CHI-PepTalk.com
&+,3HS7DONFRP

STATEMENT OF OWNERSHIP, MANAGEMENT, AND CIRCULATION

(Required by 39 U.S.C. 3685) for THE SCIENTIST (ISSN 00890-3670) Filed on September 12, 2023, published quarterly at 1000 N West St Suite 1200
Wilmington, DE 19801. The number of issues published annually is 4. The annual individual subscription price is $39.95. The general business offices of
the publisher are 334 King St, Unit 2 Midland, ON, Canada L4R 3M8. The name and address of the Publisher is Robert S. D’Angelo, LabX Media Group,
1000 N West St Suite 1200 Wilmington, DE 19801. The name and address of the Editor is Meenakshi Prabhune, LabX Media Group, 1000 N West St Suite
1200 Wilmington, DE 19801. THE SCIENTIST is owned by LabX Media Group/Bob Kafato, 334 King St, Unit 2 Midland, ON, Canada L4R 3M8. The known
bondholders, mortgagers and other security holders owning or holding 1 percent or more of the total amount of bonds, mortgages, or other securities are
none. Publication Title: THE SCIENTIST. The issue date for circulation data below (actual): Fall 2023. The average number of copies of each issue during the
preceding 12 months are: (A) Total number of copies printed: 23,094. (B1) Paid/Requested outside-county mail subscriptions stated on form 3541: 16,851.
(B2) Paid in-county subscriptions stated on form 3541: none. (B3) Sales through dealers and carriers, street vendors, counter sales, and other non-USPS paid
distribution: 236. (B4) Other classes mailed through the USPS: none. (C) Total paid and/or requested circulation: 17,087. (D1) Outside county nonrequested
copies stated on PS form 3541: 5.504. (D2) In-county nonrequested copies stated on PS Form 3541: none. (D3) Nonrequested copies Distributed thought
the USPS: none. (D4) Nonrequested distribution outside the mail: 175. (E) Total nonrequested distribution: 5,679. (F) Total distribution: 22,766. (G) Copies
not distributed: 328. (H) Total: 23,094. (I) Percent paid and/or requested circulation: 75.06%. The actual number of copies of single issue published nearest
to filing date are: (A) Total number of copies printed 20,063. (B1) Paid/Requested outsidecounty mail subscriptions stated on form 3541: 14,084. (B2)
Paid in-county subscriptions stated on form 3541: none. (B3) Sales through dealers and carriers, street vendors, counter sales, and other non-USPS paid
distribution: 186. (B4) Other classes mailed through the USPS: none. (C) Total paid and/or requested circulation: 14,270. (D1) Outside county nonrequested
copies stated on US Form 3541: 5,269. (D2) In-county nonrequested copies stated on PS Form 3541: none. (D3) Nonrequested copies Distributed thought
the USPS: none. (D4) Nonrequested distribution outside the mail: 200 (E) Total nonrequested distribution: 5,469 (F) Total distribution: 19,739. (G) Copies
not distributed: 324. (H) Total: 20,063. (I) Percent paid and/or requested circulation: 72.29%. Electronic Copy Circulation. The average number of copies of
each issue during the preceding 12 months are: (A) Requested and paid electronic copies: 29,071. (B) Total requested and paid print copies + requested/paid
electronic copies: 46,148 (C) Total requested copy distribution + requested/paid electronic copies: 51,837. (D) Percent paid and/or requested circulation
(both print & electronic copies): 89%. The actual number of copies of single issue published nearest to filing date are: (A) Requested and paid electronic
copies: 29,082. (B) Total requested and paid print copies + requested/paid electronic copies: 43,352. (C) Total requested copy distribution + requested/paid
electronic copies: 48,821. (D) Percent paid and/orrequested circulation (both print & electronic copies): 89%. I certify that all information on this statement is
true and complete: Robert S, D’Angelo, Publisher September 19, 2023.
75 YEARS OF SCIENTIFIC
BREAKTHROUGHS.
PITTCON IS A CATALYST OF SCIENTIFIC ADVANCEMENT for you,
your research, your career, your organization, and together, our
world. Our aim is to provide you with unparalleled access to the
latest advances in laboratory science, to the instrumentation
enhancing your work, and to an international assembly of
scientists experimenting, discovering, and innovating
throughout the foremost areas of focus.

LEADING IN THE LAB STARTS AT PITTCON.ORG

San Diego, CA, USA | February 24–28, 2024

From Eye to Insight

The world’s first imaging Microhub. Mica

More than a highly automated microscope, Mica unites widefield and confocal imaging in a sample protecting, incubating environment. With the
simple push of a button, you have everything you need - all in one place - to supercharge fluorescence imaging workflows and get meaningful
scientific results faster.

Simultaneous 4-color widefield, confocal resolution, AI supported analysis. All in one place.

Unified imaging modalities Point scanning confocal Mica is an incubator

Mica unifies transmitted and fluorescence
light imaging modalities such as IMC,
THUNDER and LIGHTNING in one Microhub –
for both fixed and living specimens.
Obtain highest resolution in all 3 dimensions
with point scanning confocal including optical
sectioning. The pinhole physically blocks
out-of-focus light yielding the best axial
resolution and is particularly suitable for 3D
imaging of thick samples.
The entire encapsulated inner sample space
can be climate controlled (temperature, CO2
and humidity regulation) and offers ideal
conditions for short and long-term live cell
observation.

www.leica-microsystems.com

MC-0006441. Copyright © 2023 Leica Microsystems, Deerfield, IL, USA. Subject to modifications. LEICA and the Leica Logo are trademarks of Leica Microsystems IR GmbH registered in Europe, the United
States and other countries.