The paradigm of differential

gene expression:
Epigenetic Reprogramming
Bui Hong Thuy, Ph.D.
Associated Professor
School of
Biotechnology, International
University
Email: bhthuy@hcmiu.edu.vn
1
 The question of differentiation:
A single cell, the fertilized egg, give rise to hundreds of
differentiated cell types: life cycles
Haploid gamete
(n)

Meiosis Sperm Fertilization

Ovary
Testis Egg
(Oocyte)

Multicellular diploid
adults cell (Number Mitosis & Development
of 2
chromosomes = 2n) B
Whether every cell of an organism had the
same set of genes

Neuron

2-cell embryo Blood cells

Fertilized egg
Blastocyst
(Zygote)

Liver cell

Answer: Yes, every cell of an organism had the same

set of genes (genome) 3
Chromatin Structure and DNA Packing

4
 Chromatin Structure and DNA Packing

1400 nm 300 nm 30 nm Histone H1

700 nm

Histones

Metaphase Nucleosome
chromosom
e

5
 Structure of the nucleosome core
1. Anatomy of the gene:
Nucleosome: A basic unit of chromatin structure. It is
an octamer of histone proteins (H2A, H2B, H3, and H4) wrapped
with two loops containing about 140 base pair of DNA.

Histone
octamer

H1
histones

Nucleosome structure DNA “wrap”

H3 H4 Linker DNA
H2A H2B
6
 Structure of chromatin
Histone DNA
octamer
H1

Chromatin: A complex of
DNA and protein called
Chromatin.

7
 Gene Expression
transcription translation
DNA mRNA Protein

8
Genes expression

Genes expression
9
 Differential gene expression
Question?
If the genome
in all somatic cells within an organism is
the same, how do the cell become
different
All of thefrom one
cell in theanother.
body contains the
Neuron Blood cells Liver cell
genes for hemoglobin and insulin
protein, how come hemoglobin
proteins
Answer: are made only in red cell.
Every cell nucleus contains the complete genome established in the
zygote. The DNAs of all differentiated cells are identical.
The unused genes in differentiated cell are not destroyed or
mutated, but retain the potential for being express.
Only a small percentage of the genome is expressed in each cell,
and a portion of the RNA synthesized in each cell is specific for
that
cell. 10
 Differential gene expression
• Different cell types make different sets of proteins, even
when their genomes are identical.
• Different cell types use different subsets of these genes.
• Developmental genetics is the discipline that examines how
the genotype is transformed into the phenotype.
• Differential gene transcription, regulating which of the
nuclear genes are transcribed into RNA.
• Selective nuclear RNA processing, regulating which of the
transcribed RNAs (or which parts of such a nuclear RNA) enter
into the cytoplasm to become messenger RNAs.
• Selective messenger RNA translation, regulating which of
the mRNAs in the cytoplasm become translated into proteins.
• Differential protein modification, regulating which
proteins are allowed to remain or function in the cell
11
The genetic core of
development:
Cloning animals
Bui Hong Thuy, Ph.D.
Associated Professor
School of
Biotechnology, International
University 12
Email: bhthuy@hcmiu.edu.vn
Nucleus or cytoplasm: which controls
heredity?

Somatic nuclear transfer

Question?
Wild-type Albino parent cell nuclei differ from others in
Is it possible that some differential
Recipient donor cells their ability to direct development?
eggs

Answer:
Nucleus are responsible for the
development of inherited
characters.
(Gurdon et al. 1975)

1962
13
Reprogramming of a differentiated cell
Question?

differentiated cells can

become a undifferentiated
cell or not?

Answer:

es, a differentiated cell can

reprogramming to become
a undifferentiated cell.

hus, a single differentiated

cell nucleus still retained
incredible potencies.
14
 Procedure of Nuclear Transfer in
Mice
Oocyte
donor Nuclear Activation with
Oocyte transfer CB for 6 hrs
collectio
n
Enucleation

Subsequently Embryo
culture for 1-2 culture
hr
Donor cells Embry
collection o
Surrogate transfe
mother Embryo
r
culture
Caesarian section at
Remove
Somatic donor cell
19.5dpc Bl
cell cytoplasm
nucleus
donor Cloned mice ntES15ce
Cloning animals

Dolly

1996 1997 1998 2000 2000 2002

2002 2003 2003 2005 2009

16
 Introduction
The low
success rate in cloning animal
is believed to be associated with:
Abnormal epigenetic modifications
(Histone hypermethylation and hypoacetylation)
-K9NH
Ac 3+Me
Nucleosome P S10-
DNA
-K14 Ac

-K18 Ac

H2B H2A
H4 H3 P S28- -K27 Me

DNA
H3
Octameric
17
histone core
 Materials and Methods

Mature oocyte
(M II)
NH3+

P S10- -K9 Ac Me

-K14 Ac
First polar body Somatic nucleus (SN) -K18 Ac

MII
P S28- -K27 Me

0- 2 h
Cultu SN H3
Somatic injection re
Chromatin remodeling
in Pig MII oocyte Histone modifications18
Differential timing of deacetylation of histone H3-K9
in somatic nuclear after injected into mature oocyte
0h 0.5 h 1h 1.5 h 2h
DNA
Ac-H3-K9
Lamin A/C

19
19
Differential timing of deacetylation of histone H3-K14
in somatic nuclear after injected into mature oocyte
0h 0.5 h 1h 1.5 h 2h
DNA
Ac-H3-K14
Lamin A/C

20
20
 Expression of Me-H3-K9 in somatic
nuclei after injected into MII oocytes
0h 0.5 h 1 h2 h

M II
M II
1st pb
1st 1st pb
1st M II pb
pb M II

SN SN SN SN

Histone H3 methylation of somatic cell nuclei did not

show a significant change after injection to the oocytes
Hong-Thuy Bui et al.
Conclusion

1. Somatic nuclei reach to metaphase

stage and become deacetylation of
histone H3.

2. Methylation of H3-K9 in somatic nuclei

was maintained after injected into MII
oocytes.

Bui et al. Reproduction 2006

22
Effect of Trichostatin A on chromatin
remodeling, histone modifications,
DNA replication and transcriptional
activity in cloned mouse embryos
(Hong-Thuy Bui et al. 2010) 23
Introduction
Our group found that inhibition of histone deacetylase
by Trichostatin A (TSA) after somatic cell nuclear
transfer (SCNT) increase the development of mouse
cloning (Kishigami et al. 2006 ）
We believe that initial somatic cell chromatin
remodeling plays an important role in
genomic reprogramming NH3+

-K9 Ac Me

The effect of TSA on: P S10-

-K14
*Chromatin remodeling
Ac

-K18
*Histone modifications
Ac

*DNA replication -K27 Me

*Transcriptional activity P S28-

in early SCNT mouse embryos.

H3 24
 B6D2F1
mature
B6D2F1
cumulus
Materials and
oocyte cells Methods
1-2
hours

Enucleation Nuclear transfer Activated for 6 h by Sr+CB &

(Cloned •with TSA 50 nM TSA+)
•without TSA (TSA–)
embryo)
Culture for 4 h
(TSA + or Culture
TSA-)
1-cell 2-cell
1. DNA replication: Label 5-bromo-deoxyuridine (10 M BrdU)
2. Chromosome decondensation: Phosphorylation of H3 at Serine
28
3. Histone modifications: Acetylation & Methylation of histone H3
4. Transcriptional activity: Detection of nascent RNA
 Effect of TSA on DNA replication in 1-cell
SCNT embryos

4h 5h 6h 7h 8h 9h 10 h
NT+TSA NT–TSA

*Mouse monoclonal anti-BrdU (Roche)

1. TSA treatment promoted DNA replication in

SCNT embryos

(Hong-Thuy Bui et al. Biology of Reproduction, 2010)6

Effect of TSA on

Number of embryos
100%

+TSA
90%
80%

Chromosome 0/
70%
60%
10

Decondensation
50%

–TSA
40%
30%
20%
10%
0%
10 11 12 13 14 15 16 h

DNA P-H3-S28 Lamin DNA P-H3-S28 Lamin Decondense

chromosome
10 h

Condensing
chromosome
Condensed
chromosome
12 h

2. TSA increased
14 h

chromosome
decondensation
16 h

in SCNT embryos
18 h

27
NT–TSA NT+TSA (Hong
 Effects of TSA on Nuclear Volume and Histone
H3 acetylation at lysine 9 in SCNT embryos
6h 8h 10 h 12 h Ac-H3-K9 & Lamin B
Nuclear volume125

After activation
120

6h
115

110

10 h
105

100
95

90
NT–TSA NT+TSA
NT-TSA embryos

Intensity of Ac-H3-K9
NT+TSA embryos 6h 10 h
100

80

3. TSA 60

increased Nuclear volume and 40

Histone acetylation in pronucleus of

20
0

SCNT embryos 28
(Hon
 Expression of Dime-H3-K4 in ICSI and NT embryos
without (NT–TSA) and with TSA (NT+TSA)
treatment

Intensity of Dime-H3-K4
ICSI NT-TSA NT+TSA 120

♀
Dime-H3-K4 DAPI

100 c c

♂ 80

One-cell
d
a
60 a
40 b

20

One-cell
Two-cell
ICSI embryos
NT-TSA embryos
Dime-H3-K4 DAPI

NT+TSA embryos
Two-cell

4. TSA increased Dime-H3-

K4 in SCNT similar to ICSI
embryos
29
(Hong
 Expression of Trime-H3-K9 in ICSI and NT embryos
without (NT–TSA) and with TSA (NT+TSA)
treatment
ICSI NT-TSA NT+TSA

Intensity of Trime-H3-K9
40
b
b
DAPI

30
c c

One-cell
♂ 20
♀ a
a
Trime-H3-K9

10

♀ ♂ One-cell Two-cell
ICSI embryos
NT-TSA embryos
Trime-H3-K9 DAPI

Two-cell NT+TSA embryos

5. TSA reduced Trime-

♂
H3-K9 in SCNT
♀ ♂
embryos
♀ 30
(Ho
Nascent RNA expression in ICSI and NT embryos without
(NT–TSA) and with TSA (NT+TSA) treatment
DAPI Br-UTP

Number of embryos (%)

ICSI

100

80

60

40
NT

20
0

High Medium Low

Bright DAPI Br-UTP Ac-H3-K9 ICSI embryos

NT-TSA embryos
NT+TSA
embryos

6. TSA increased nascent RNA production and

normal chromatin morphology in SCNT embryos
31
(Hong-Thuy Bui et al. BOR, 2010)
 Conclusion
The initial somatic chromatin
remodeling plays a key role in genomic
reprogramming and important for the
further development of cloned embryos.

Biology of Reproduction 2011

Histone Deacetylase (HDAC) Inhibition
Improves Activation of Ribosomal RNA Genes
and Embryonic Nucleolar Reprogramming in
Cloned Mouse Embryos
Hong-Thuy Bui et al. 2011
32
 Introduction

The nucleolus is
a complex nuclear area where ribosomal RNA (rRNA) are
synthesized and contain many nucleolar proteins such as
Embryonic development Introduction

The nucleolus is present in the nucleus of early

mammalian embryos, which structure support active
rRNA genes.
34
Objective
1. Examine the effect of Scriptaid on the onset of rRNA
synthesis in cloned embryos by using highly sensitive
fluorescence in situ hybridization (FISH) with probes
complementary to mouse rDNA.
2. Examine reactivation of nucleolar protein and rRNA
genes by using immunofluorescent detection of proteins
and 5-bromouridine 5’-triphosphate (BrUTP)-labeled
nascent rRNAtranscripts.
*Assessment of rRNAsynthesis
*Allocation of Nucleolar Proteins
- rRNAsynthesis (e.g., upstream binding factor [UBF] )
- rRNAprocessing (e.g., fibrillarin and nucleophosmin/B23)
35
 B6D2F1
Materials
B6D2F1
mature
oocyte
cumulus
cells
and
1-2 Methods
hours

Enucleation Nuclear transfer Activated by Sr+CB &

(Cloned treatment for 6 h:
•with Scr (250 nM Scr +)
embryo) •without Scr (Src -)

Culture for 4 h Culture

(Scr + or Scr-)

1-cell
2-cell
36
 Materials and
Experiment 1 Methods

In situ hybridization
Embryos spread
Relation of rRNA and
Transcriptional activity

Early 2-cell Compare rRNA activity

(18h) of ICSI, NT-Scr and
Late 2-cell (32h) NT+Scr

37
(Hon
 Analysis of rRNA gene expression by FISH of ICSI (A, B)
and
SCNT (C, D, E) embryos at
*C57BL/6J inbred mice contain four nucleolar the early and late
ICSI 2-cell stages
A B
organizing region-bearing chromosomes per
haploid set.
*F1 (C57BL/6J x CBA/Ca hybrid) embryos
* * *
contain seven rDNA clusters in diploid
metaphase chromosome * *
(three rDNA from paternal CBA/Ca mice)
*
Silent transcription Competent transcription

C * D SCNT E *
* *
* *
*
*
* * * *
Silent transcription Competent
transcription
The nuclei containing five or more activated rDNA were classified as competent
transcription; those containing four or fewer were classified as silent transcription.
The merged images indicate FISH signals (rDNA): red and DAPI-labeled DNA :
blue
38
 Embryos were analyzed by FISH to detect rRNA
transcriptional activity
No. (%) of two-cell embryos with
Groupstranscriptions
Cell stage
Silent Asymmetric Competent
transcription transcription transcription
ICSI
Early 2-cell 49 (100)a 0 ( 0)a 0 ( 0)a
Late 2-cell 4 ( 9) b 0 ( 0) a 41 ( 91)b
NT-Scr
Early 2-cell 40 (100)a 0 ( 0)a 0 ( 0)a

Late 2-cell 23 ( 61) c 13 ( 34)b 2 ( 5) a

NT+Scr
Early 2-cell 44 (100)a 0 ( 0) a 0 ( 0)a
Late 2-cell 24 ( 60) c 6 ( 15)c 10 ( 24) c

Proportion of embryos expressing rDNA transcriptional activity at the early

two-cell and late two-cell stages in ICSI-generated embryos, SCNT embryos
without Scriptaid (NT-Scr), and SCNT embryos with Scriptaid treatment
39
(NT+Scr).
Conclusion 1
The results show that in the late 2-cell stage, a number of
SCNT embryos initiated transcriptional activation while
having one blastomere showing inactivated rRNA
transcription and another blastomere showing activated
rRNA transcription and despite both nuclei being in interphase.

Scriptaid Promotes Onset of rRNA Synthesis in

Cloned Embryos

40
Experiment 2 Materials
＋
and
ー Methods
Electrical Immunostaining
permeabilization •Transcriptional activity
10 mM BrUTP
(Nascent rRNA) Anti-
Alpha amanitin
bromodeoxyuridine antibody,
*ICSI embryos Culture in CZB 1 h Alexa 568
*NT-Scr embryos Alpha amanitin
*NT+Scr emryos •Nucleolar proteins
2-cell (18 h, 24 h, 32 h) *Upstream binding factor
4-cell (48 h)
8-cell (62 h) *Fibrillarin
Blastocyst (84 h) *Nucleophosmin

α-amanitin inhibit polymerase II and III but not inhibit polymerase I. Br-
UTP signals will associated with the nucleolus surface
41
(Hong-Thuy Bui et al. BOR, 2011)
 Inactivated
A) a Merge b rRNA B) a b

rRNA
Inactivated

UBF
** *

Activated & Inactivated

c d
c d

Fibrillarin
rRNA
*

Activated
*

* e f

Activated

B23
rRNA
FIG. 2. Expression of rRNA and nucleolar proteins at the early,
middle,
and late 2-cell stages
A (a&b) Nucleus of an early 2-cell embryo indicates inactivated rRNA with
BrUTP signals
condensed into small distinct clusters.
(c&d) Nucleus of a late 2-cell embryo indicates activated rRNA with decondensed
inactivated and activated rRNA, and activated rRNA. 42
BrUTP
(Hong
 A) B)
DNA UBF Merge DNA Fibrillarin Merge
a b c a b c
Early 2-cell

d e f d e f
Late 2-cell

*
**

g h i g h i
4-cell

*
*

Distribution of nucleolar proteins in early 2-cell, late 2-cell, and 4-

cell embryos
A) Embryos were labeled for upstream binding factor (UBF). In the early 2-cell stage with inactivated
rRNA transcription (a–c), UBF was detected weakly in small cytoplasmic foci, some of them near
NPBs (arrows). At the late 2-cell and 4-cell stages (with activated rRNA transcription; (d–f) and (g–
i), respectively), UBF emerged to form large foci around NPBs.
B) Embryos were labeled for Fibrillarin. At the early 2-cell stage (inactivated rRNA transcription; a–c),
fibrillarin was detected in numerous small foci located at the periphery of NPBs. At the late 2-cell
and 4-cell stages (activated rRNA transcription; d–f and g–i, respectively), fibrillarin showed a
complex distribution with accumulation and migration around NPBs. 43
Distribution of nucleophosmin in ICSI (A) and SCNT (B)
embryos
Pronucleus Early 2-cell
duringLatepreimplantation.
2-cell 4-cell 8-cell Blastocyst
c e g i k
DNA

a
Nucleophosmin

b d f h j l

a c e g i k
DNA
Nucleophosmin

b d f h j l

44
 Conclusion 2
The asynchronous transcription induced a delay of
one cell cycle in SCNT embryo activation of
functional nucleoli.

Scriptaid Promotes Normal Onset of Zygotic

Gene Activation (ZGA) Concomitant with
Nucleolar Reactivation in Cloned Embryos.

45
 Summary
The findings suggest that Scriptaid can overcome failure in
the timely onset of embryonic gene transcription. Promotion of
ribosomal RNA gene activation and nucleolar protein
allocation during the early phase of ZGA.

This demonstrate that rDNA transcription is a useful

marker for monitoring the activation of embryonic genes, a
principal event of early embryonic development.

46
Determining the Function of
Genes during Development:
Transgenic Animals
Bui Hong Thuy, Ph.D.
Associated Professor
School of
Biotechnology, International
University
Email: bhthuy@hcmiu.edu.vn
47
Why Clone Mammals is important?
Given that we already knew from amphibian studies in the 1960s that
nuclei were pluripotent, why clone mammals? Many of the reasons
are medical and commercial, and there are good reasons why these
techniques were first developed by pharmaceutical companies rather
than at universities.
• Cloning is of interest to some developmental biologists who
study the relationships between the nucleus and cytoplasm
during fertilization or who study aging (and the loss of
totipotency that appears to accompany it), but cloned
mammals are of special interest to those people concerned
with protein pharmaceuticals.
• Protein drugs such as human insulin, protease inhibitor, and
clotting factors are difficult to manufacture. Due to
immunological rejection problems, the human proteins are
usually much better tolerated by patients than proteins from
other animals. So the problem becomes how to obtain large
amounts of the human protein? 48
 Overview
One of the most efficient ways of
producing these proteins is to insert the human genes
encoding them into the oocyte DNA of sheep, goats, or cows.
Animals containing a gene from another individual (often
of a different species) a transgene are called transgenic
animals.
A transgenic female sheep or cow might
not only contain the gene for the human protein, but might
also be able to express the gene in her mammary tissue and
thereby secrete the protein in her milk. Thus, shortly after the
announcement of Dolly, the same laboratory announced the
birth of Polly (Schnieke et al. 1997). Polly was cloned from
transgenic fetal sheep. Fibroblasts that contained the gene for
human clotting factor IX, a gene whose function is deficient in
hereditary hemophilia.
 Overview
* Producing transgenic sheep, cows, or
goats is not an efficient undertaking. Only 20% of the treated
eggs survive the technique. Of these, only about 5% express the
human gene. And of those transgenic animals expressing the
human gene, only half are female, and only a small percentage
of these actually secrete a high level of the protein into their milk.
(And it often takes years for them to first produce milk).
* Moreover, after several years of milk
production, they die, and their offspring are usually not as
good at secreting the human protein as the originals.
* Cloning would enable
pharmaceutical companies to make numerous copies of such an
"elite transgenic animal," all of which should produce high yields
of the human protein in their milk.
* The medical importance of such a
technology would be great, since such proteins could become 50
much cheaper for the patients who need them for survival.
 Introduction
Application of transgenic
animals
Since the first gene transfers into
mice were successfully executed in 1980,
transgenic animals have allowed researchers
to observe experimentally the roles of genes
in development, physiology and disease.
Recently, transgenic animals are
applied
for the purposes of the production of
pharmaceuticals (Human protein drug) . 51
 What is Transgenic Animal?
A transgenic animal is one that
carries a foreign gene that has been
deliberately inserted into its genome.
In addition to a structural gene, the
foreign gene (DNA) usually includes other
sequences to enable it:
• to be incorporated into the DNA of the host.
• to be expressed correctly by the cells of the
host.
52
The goals of Transgenic Animals
• In medical research: identify the functions of specific factors
in complex homeostatic systems through over- or under-
expression of a modified gene (the inserted transgene).
• In molecular biology: analysis of the regulation of gene
expression makes use of the evaluation of a specific genetic
change at the level of the whole animal (for experiments of
the phenotype effects of transgene expression).
• In the pharmaceutical industry: targeted production of
pharmaceutical proteins, drug production and product
efficacy testing.
• Transgenic animals can be used as bioreactors for the
p ro d u c t i o n of re c o m b i n a n t proteins that h a v e
pharmaceutical applications. Such animals are sometimes
called "Pharm Animals".
53
 Methods of Producing Transgenic Animals via
Micromanipulator Technique
The three principal methods used for the creation
of transgenic animals are
1. DNA microinjection.
- Injection into pronuclear formation
-Injection with sperm via ICSI.
2. Embryonic stem cell-mediated gene transfer
*Blastocyst injection.
- Diploid blastocyst
- Tetraploid blastocyst
*8-cells embryo injection
3. Cloning techniques. 54
 Methods of Producing Transgenic Animals via
Micromanipulator Technique
The three principal methods used for the creation
of transgenic animals are
1. DNA microinjection.
- Injection into pronucleus
-Injection with sperm via ICSI.
2. Embryonic stem cell-mediated gene transfer
*Blastocyst injection.
- Diploid blastocyst
- Tetraploid blastocyst
*8-cells embryo injection
3. Cloning techniques. 55
Micromanipulator system

1. ICSI
2. Blastocyst injection
3. Enucleation
4. Nuclear transfer
5. Produce transgenic Animal56
Piezo-actuated micromanipulator

Piezo-actuated
micromanipulator

57
 DNA Injection into pronucleus
Unsuccessful

Successful

Microinjectio
n of DNA into
pronuclear formation 58
Pronuclear injection in pig
Pig zygote
Centrifugation
6,500 g for 10 min

GFP transgenic pig Pronuclear injection

Embryo
transfer

59
 DNA microinjection via ICSI
1. Disruption of sperm head membranes

A. Fresh sperm: Intact

membranes

B. Whose membranes had been

disrupted by Triton X-100 (B),
C. Whose membranes had been
Disrupted by freeze-thawing.
C. Whose membranes had been
Disrupted by freeze-drying.
a b c d
60
 Materials and Methods

DNA
ICSI

Mixtur
20 % offspring
e of DNA & expressing the
Sperm membranes membranes
had been disrupted disrupted
integrated
spermatozoa transgene.

Mammalian Transgenesis by Intracytoplasmic Sperm Injection

Anthony C. F. Perry, 1* Teruhiko Wakayama, 1 Hidefumi Kishikawa, 1 Tsuyoshi Kasai, 1 Masaru Okabe, 2 Yutaka
61
Toyoda
 DNA microinjection via ICSI
Transgenic embryos
produced by single shot double
transgenesis. Oocytes were microinjected
with spermatozoa that had
been preincubated with DNA.

A. The same embryos are shown (×400) after

3.5 days
viewed by Hoffman modulation contrast
microscopy unstained
B. for GFP expression under long-wavelength
(480 nm) UV light,

High GFP expression at the

morula and blastocyst stage after injection of GFP DNA
mixed with freeze-dry sperm visa ICSI technique
 DNA microinjection via ICSI
Sperm treatment Total pups Positive with
GFP (green)
Freeze-dry sperm 14 3 (21%)
Freeze-thaw 12 2 (16%)
Triton X-100 treated sperm 31 6 (19%)
Intact sperm (control) 42 0 (00%)

Disruption of sperm GFP+ GFP-

head membranes are

very important for
the success of
transgenic animal
visa ICSI
63
 Methods of Producing Transgenic Animals via
Micromanipulator Technique
The three principal methods used for the creation
of transgenic animals are
1. DNA microinjection.
- Injection into pronucleus
-Injection with sperm via ICSI.
2. Embryonic stem cell-mediated gene transfer
- 8-cells embryo injection
- Diploid blastocyst
- Tetraploid blastocyst

3. Cloning techniques. 64
 Materials and Methods
* Transfection: Incorporated directly into cells by incubating the cells with DNA.
* Electroporation : Using a high-voltage pulse the DNA into the cells

ES cells DNA
Transfection
Electroporati
Positive, negative selection
blastocyst on

Negative
Positive
Diploid Pig (GFP-)
(GFP+) Diploid expanded
8-cell Mouse blastocyst
injection injection

Proliferation of
ES cells
carrying targeted gene
65
Materials Proliferation of ES cells
and carrying targeted gene
Methods

Surrogate
Fertilized 8-cell
mother
embryo

Embryo
transfer
Fertilized Blastocyst

Tetraploid Blastocyst 66
How to inject ES cells into 8-cell and blastocyst

Blastocyte injection 8-cells injection

Using piezo action Using XYclone

laser
Note: Only small ES cells are selected for injection
Optimal number of ES cells is 25-
30/blastocyst 67
 Diploid blastocyst injection
Injection of gene targeted ES cells in to normal
fertilized embryo at the expanded blastocyst
stage

Diploid blastocyst
B6D2F1 mouse injection

Diploid Chimaeric mouse

ICR mouse
Diploid blastocyst injection to produce chimaeric mouse
68
ES cells from B6D2F2 injected into blastocyst received from ICR
 Tetraploid blastocyst injection
Fetus was developed
from ES cells
ES cells

Injection

Tetraploid Placenta was developed

blastocyst from tetraploid embryo
69
 Methods of Producing Transgenic Animals via
Micromanipulator Technique
The three principal methods used for the creation
of transgenic animals are
1. DNA microinjection.
- Injection into pronucleus
-Injection with sperm via ICSI.
2. Embryonic stem cell-mediated gene transfer
*Blastocyst injection.
- Diploid blastocyst
- Tetraploid blastocyst
*8-cells embryo injection
3. Cloning techniques. 70
 Procedure of Nuclear Transfer in
Mice
Oocyte
donor Nuclear Activation with
Oocyte transfer CB for 6 hrs
collectio
n
Enucleation

Subsequently Embryo
culture for 1-2 culture
hr
Embry
o
Surrogate transfe
mother Embryo
r
culture
ES cells carrying Caesarian section at
Remove 19.5dpc
targeted gene donor cell Bl
cytoplasm
71
Cloned mice ntES
Genetically Modified Animals
and Medical Applications
Bui Hong Thuy, Ph.D.
Associated Professor
School of
Biotechnology, International
University
Email: bhthuy@hcmiu.edu.vn
Part I: Produce human therapeutic
protein
Human proteins Positive, Dairy cow selection
negative cells
producing genes DNA
Transfection
or Electroporation Positive
Negativ
e (GFP+)
(GFP-)

Cloned
transgeni
c
embryo
Cloning animal
Embryos
transfer

Human Transgenic cow

proteins 73
 Great
business?
Therapeutic Proteins
AAT (alpha-1-antitrysin)
Animal
Cow
$/animal/year

150.000 USD/cow/year
tPA (Tissue plasminogen Cow
700.000 USD/cow/year
activator, chống đông máu)
Factor VIII or IX or (Blood Cow
200.000 -300.000
clotting factors, điều trị chống
chảy máu). USD/cow/year

Lactoferrin 25.000 USD/cow/year

CFTR (Cystic fibrosis Cow
800.000 USD/cow/year
transmembrane conductance
regulator) Supper dairy cow
Human Protein C (Chống đông Cow 10 milions
máu)
Cow
USD/cow/year 200.000.000
Fibrinogen (điều trị vết thương) 400.000 USD/cow/year
Glutamic acid decarboxylase Cow 200.000 USD/cow/year USD/cow
(Điều trị tiểu đường type 1)
Anpha-lactalbumin (điều trị Cow 500.000 USD/cow/year
nhiễm trùng)
74
Researchers rear hens that lay drug-filled eggs
The National Institute of Advanced Industrial Science
and Technology (AIST) in Japan has succeeded in
making hens lay eggs that contain a pharmaceutical
agent that can be used to treat such diseases as cancer
and hepatitis (Oct. 8th 2017)
Genome editing to
produce Interferon beta,
a type of protein related
to the immune system

In late July, 2017 at the

company’s breeding facility
in Hokkaido, female
chickens with modified
genes laid eggs, which the
researchers confirmed
contained interferon beta
in the whites of the eggs 75
 An Introduction: Generalities over
Genetic Engineering of Animals to
Create Pharmaceutical Drugs

There are many pharmaceutical drugs used today to treat illnesses

that have come from genetically modified animals, or transgenic animals.
This is slowly becoming a common way to create drugs that we otherwise
could not create due to lack of a certain modification we could find
elsewhere to create the drug or costly expenses. Expensive meaning it
could cost the government millions of dollars because of the lack of
effectiveness without animal modification and would also be expensive for
patients.

“Many therapeutic proteins for the treatment of human

disease require animal cell specific modifications to be effective,
and at the present time they are almost all produced in
cell-based bioreactors” 76
mammalian
 IMPORTANT TERMS
TO
Recombinant DNA REMEMBER
Fixed DNA that has combined fragments of
DNA put together in a laboratory for
a desired use.
Transformation The transformation of the DNA being fixed
by the recombinant, which genetically
modifies the animal.
Genetically modified, engineered, or The combination of both the animals
transgenic animal own genes and the recombinant DNA
produces an animal with a newly made
co m b in a ti o n of genes to p ro d u c e a
specific type of DNA.

Gene Vector Plasmid or virus

Pharmaceutical Drugs
Therapeutic proteins
Drugs given to patients to treat an illness or
disease.
Protein in the drug which targets the illness
or disease in order to cure it.
77
 The Basic Process of Genetic Engineering
Many animals are being used to produce
pharmaceutical drugs, mainly milk and egg
producing animals such as chicken, goats, and
cows. The common way to genetically modify
animals is a
certain process performed in the laboratory

1.The gene of interest is isolated on a strand of

DNA 2.DNA is cut at specific points by restriction
enzymes. The enzymes recognize certain
sequences of bases on the DNA strand and cut
where those sequences appear.
3.The cut DNA joins with a vector, which may be a
virus or part of a bacterial cell called a plasmid.
The vector carries the gene of interest into the
organism that will produce the protein.
4.Transformation occurs when the gene carried by
the vector is incorporated into the DNA of another
organism where it initiates the action desired

it states that drugs can be produced also in E-coli bacteria or plants. However, with animals, it is much
78
more efficient.
 The Basic Process of Genetic
Engineering

1. Plasmid removed from E-coli

cell
2. The Plasmid is opened by a
special enzyme
3. DNA coding for human drug is
inserted in the opened Plasmid.
4. Recombination – Plasmid closed
by another special enzyme
5. Introduction of Recombined
Plasmid into E-coli host cell.
6. Host cell divides into new cells
identical to the original. The
implanted DNA induces the cell to
produce the drug.

79
Examples of Drugs Made from
Genetic Engineering of Animals

•ATryn
•Insulin
•Lactoferrin (Breast Milk
Supplement)
•Erythopoietin
80
 ATryn
DRUG ANIMAL USED GENETIC WHO/WHERE
MODIFICATION PRODUCED

ATryn Goats Genetically “In 2006, there are

modifying the 57 goats being
goats to produce engineered in
milk with the Charlton,
anti- Massachusetts to
blood thinning produce the anti-
protein, blood clotting
Antithrombin, drugs called
from their ATryn in their
mammary glands milk”.
to fight blood
clotting diseases.
81
 ATryn
• Goats are one of the first animals to be used in
the
• genetic
Biotech engineering
Therapeuticsprocess.
Currently (GTC) owns a farm
in
• In Charlton,
2006, 57Massachusetts.
goats were being used to make the
anti-
blood clotting protein,
• Antithrombin Antithrombin,
is produced on this farm.
from the mammary glands
of
• In transgenic goats,Biotherapeutics
2009, GTC- and harvested Ovation
from their milk.
Pharamceuticals
Inc. announced that the Food and Drug Administration
(FDA) had approved ATryn (Antithrombin Recombiant)
for the prevention of blood clotting (đông máu) in
Antithrombin deficient patients.
• Choosing to genetically engineer goats to produce
the drug is an efficient method and useful because
Antithrombin is hard to come by or is simply not
available.
• After furthur clinical trials, this is expected to be
available
in the pharamacy by 2012. 82
 FDA clears GTC's ATryn, first US-approved drug
made from a transgenic animal
* ATryn is the first ever transgenically-produced
therapeutic protein and the first recombinant antithrombin approved in
the USA. To make ATryn, scientists insert DNA for the human
antithrombin protein into a goat embryo. This embryo is implanted
into host mother. The resulting transgenic offspring are able to
produce high levels of antithrombin in their milk, which is then purified
to make the finished product that can be administered to patients by
intravenous infusion.
* The FDA's Center for Veterinary Medicine also
approved GTC's New Animal Drug Application, the first of its kind to
regulate genetically- engineered animals. This is now required for a
recombinant technology used to develop transgenic animals.
* GTC's intellectual property includes a patent in the USA
valid through 2021 for the production of any therapeutic protein in the
milk of any transgenic mammal. According to the company, this
production platform is particularly well suited to enabling cost-
effective
systems, asdevelopment of proteins
well as proteins that are that are difficult
required in largeto express in
volumes. 83
traditional recombinant production
 Lactoferrin &
Insulin
Most common from genetically modified cows
are lactoeferrin, breast milk produced from cows
in the form of a supplement, and insulin. Normal
human milk from a mother given to the baby is
nutritional, but from genetically modified cows ,
there is more of a “nutritional boost”. So it is
much healthier for the baby. Lysozome, which
fights bacteria and improves infants immune
system in their few days of life, can be found in
genetically modified cows, which has properties
of breast milk. Researchers believe human milk
from cows could be a better alternative than
baby
formula. Insulin can also be made from
84
genetically engineering cows. Insulin is more
 Erythropoietin (Epoetin Alfa or EPO)

• Many mammals are being genetically

engineered to produce Erythropoietin, like
sheep or cows. EPO is used to treat anemia or
given to patients after chemotherapy. “EPO is
a glycoprotein (protein-sugar conjugate) that
serves as the primary regulator of red blood
cells (erythrocytes) in mammals” (Ritter).
Recombinant DNA isolates the human gene
erythropoietin and is put into the mammal to
target a more modified and better version of
erythropoietin. EPO is a natural substance
produced from the kidneys in our bodies but
transporting the gene to a mammalian animal,
such as a sheep, causes a large quantity and
quality of the drug.

85
Recombinant DNA of
EPO
 • “Erythropoietin [epoetin alfa (Epogen,
Procrit)] is used in many installation-fitting
Erythropoietin clinic. The most common use is in people
(Epoetin Alfa with anemia associated with abnormal
or function (dysfunction) kidney. When the
kidneys are not functioning properly, they
EPO) produce less than normal amounts of
erythropoietin, which can lead to the
production of red blood cells are low, or
anemia. Therefore, by replacing
erythropoietin with an injection of
erythropoietin from a genetically engineered
animal, anemia associated with kidney
disease may be treated” (World of Health).

• Many athletes, such as cyclists and

runners, use EPO because the increasing
number of red blood cells can increase
oxygen capacity and produce improved
physical activity. Creating Erythropoietin
through genetic engineering, it is much
more efficient than what humans can
 Part II: Organs for human

For
Xenotransplantation
(pig’s organs to human)
Cell Reprogram. 2012
α1,3-Galactosyltransferase Deficiency
in Germ-Free Miniature Pigs Increases N-
Glycolylneuraminic Acids As the Xenoantigenic
Determinant in Pig-Human Xenotransplantation.
Park JY, Park MR, Bui HT 87
88
 Organs for human
Generation of RAT pancreas in Pdx1 knockout mice
Kobayashi et al., Cell 2010

Mouse Pdx1 -/-

(Blastocyst) Rat (ES cells)

Chimeric mouse
with rat pancreas

0ur hypothesis: Can we make

human organ from pig cells??
89
 Future: pig produce human organs?
Organ related genes
knockout cloned pig
Pig enucleated
oocyte
blastocyst
Embryo
transfer
Pig fibroblasts cells

siRNA Lentiviral
Transfection
vector

Cloned
pig with human
Pig knockout organs
pancreas, heart,
liver, kidney related
Human
iPS
?? ? ? ?
genes cells cells ? ?
? Humans?
organs
90