Creating Transgenic Animals

Transgenic - introducing a gene from one species into another species

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Creating transgenic mice

- the newly fertilized egg still has a sperm pronucleus and an egg pronucleus
- foreign DNA can randomly or specifically integrate into the genome
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- It will be present on one chromosome in all the cells of the offspring

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Creating transgenic mice

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Creating transgenic mice

- can knockout genes (delete them) and study the phenotype caused
- can modify gene in some way and put them back in and study the effect
- can alter genes to mimic human diseases and build a mouse model
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- mice are the major model system for studying human development and disease

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Producing Proteins or Drugs in Transgenic Animals

expressed in
mammary tissue

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Producing Proteins or Drugs in Transgenic Animals

2009 - antithrombin protein

(anti blood clotting drug)
approved by this method
- produced in a herd of goats
in Massachusetts
- one goat can produce as
much drug in its milk in a year
as would be purified from
90,000 blood donations

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Genetically-modified fish

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Wild type salmon

AquaBounty salmon
- 18 years and still waiting in
the US
- production was recently
approved in Canada
- still need approval for
consumption of fish
- a large number of US
stores have already
pledged not to sell it
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Making transgenic plants

- one method to make transgenic plants uses the Ti plasmid to introduce foreign DNA
- Ti plasmid is from a bacteria called Agrobacterium tumefaciens that naturally infects
plant cells and transfers DNA into them!
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Making transgenic plants

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Making transgenic plants

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About 12% of the worlds usable land is currently

planted with transgenic plants

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Producing Bt-expressing plants: pest resistance

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Producing Bt-expressing plants: pest resistance

- first used in 1996
- now more than 90% of our soybeans and
60% of our corn has been modified to
express the Bt gene

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Creating Roundup Ready plants

- farmers spend a lot of money on weedkiller to kill weeds in their
crop fields
- the active ingredient in the weedkiller Roundup is glyphosate

- glycophosate inhibits an enzyme (ESPS) in the plant chloroplast

required to make several amino acids
- unfortunately, both crop plants and weeds are sensitive to the
weedkiller

- however, there is a
bacterial ESPS enzyme
that is resistant to the
effects of glyphosate

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Creating Roundup Ready plants

- introduce into crop plants a

genetically-engineered form of
the bacterial ESPS enzyme that
is resistant to glyphosate
- the crop plants are now
Roundup ready

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Dead weeds cant become resistant some Monsanto scientist

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Golden Rice - introduced six foreign genes!

- rice is a major plant staple in large parts of the world, but it has a few

problems nutritionally

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Golden Rice - introduced six foreign genes!

1) beta-carotene is made by green vegetables and is converted into vitamin A
- vitamin A deficiency results in increased disease susceptibility and even
blindness, and increases childhood mortality
- rice cannot synthesize beta-carotene
- therefore vitamin A deficiency is widespread where rice is consumed

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Golden Rice - introduced six foreign genes!

2) iron is needed to make heme, a major component of hemoglobin, and
humans cannot make heme but must get it from their diet
- rice makes phytate, a compound that binds iron and interferes with its
absorption in the intestine
- therefore, iron deficiency (anemia) is another major problem in areas of
high rice consumption

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Golden Rice - introduced six foreign genes!

1) rice cannot synthesize beta-carotene
- engineered into rice the genes for four
enzymes needed to make beta-carotene
(two from daffodil, two from bacteria)

2) rice makes phytate, which binds iron

- engineered into rice genes to make a
bacterial enzyme that breaks down phytate,
and a bean protein that helps with iron
absorption

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Golden Rice - introduced six foreign genes!

- bad news
- you have to eat all of your daily rice as
golden rice to get the recommended
amount of beta-carotene
- after 14 years, it is still not being grown
anywhere (it is being tested in the
Phillipines)
- there is significant opposition to it by
people and governments in Asia and Africa

- http://www.npr.org/blogs/thesalt/
2013/03/07/173611461/in-a-grain-of-goldenrice-a-world-of-controversy-over-gmofoods

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general structure of a gene

this region is transcribed

- the upstream region in DNA contains information

which indicates where to start transcribing
- also when to transcribe the gene (under what conditions)

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Regulation of eukaryotic genes is more complex

- a typical eukaryotic gene is regulated by multiple

transcription factors

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Methods to study where and when a gene or protein is expressed

1) perform antibody staining - make an antibody to your

protein in rabbits or mice, purify that antibody and use it to
find where and when the protein is made using fixed cells
or tissues

- expensive, takes time, cells are fixed

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Methods to study where and when a gene or protein is expressed

2) perform in situ hybridization make a labeled probe

complementary to the mRNA and use it to find where and
when the mRNA is produced in fixed cells or tissues

- resolution is so-so, cells are fixed

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Methods to study where and when a gene or protein is expressed

3) make a reporter construct fuse the upstream region

of your gene of interest to the coding sequences for green
fluorescent protein and look where and when GFP is
expressed in living animals

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Many marine organisms show bioluminescence

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The Nobel Prize in Chemistry 2008

Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

80

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jellyfish green fluorescent protein = GFP

When blue light
shines on this
protein, it emits
green light back
81

The Nobel Prize in Chemistry 2008

Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

Can GFP be used in other organisms to make reporter constructs?

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Reporter constructs made with GFP can show

where and when a gene or protein is expressed

promoter for YFG

your favorite gene

A transcriptional reporter makes GFP whenever and wherever

the normal gene is expressed

promoter for YFG

A translational reporter makes GFP whenever and wherever

the normal protein is expressed (protein fusion)

promoter for YFG

YFG ORF
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The Nobel Prize in Chemistry 2008

Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

84

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The Nobel Prize in Chemistry 2008

Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

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Reporter constructs made with GFP can show

where and when a gene or protein is expressed

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87

Retroviral Vectors Have Been Used in Gene Therapy

- molecular biology techniques are used to introduce a human gene into a

retrovirus that infects human cells, and use the recombinant virus to carry
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the gene into the patients cells

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Retroviral vector-based gene therapy

1) the wild type copy of the gene defective in the disease is
inserted into a retroviral vector RNA genome
2) the recombinant viruses are used to infect defective cells
taken from the patient
3) the viral RNA is converted to DNA by reverse transcriptase
4) integration of the viral DNA introduces the wild type gene
into the genome of the patients cells
5) these cells are grown in tissue culture and reintroduced into
the patients body

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Retroviral vector-based gene therapy: successes

- retroviral-based somatic cell gene therapy has been used to treat
severe combined immune deficiency (SCID)
- mutations in several different genes can result in loss of both arms of the
immune system - B cells and T cells
- leaves patients completely sensitive to all infections (bubble boy)
- ADA SCID
(adenine deaminase)
- X-linked SCID
(gamma chain of IL receptor)

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Retroviral vector-based gene therapy: successes

- mutations in several different genes can result in loss of both arms of the
immune system - B cells and T cells
- leaves patients completely sensitive to all infections (bubble boy)
- ADA SCID
(adenine deaminase)
- X-linked SCID
(gamma chain of IL receptor)

1990 - patient at NIH is given the first gene therapy - viruses with a good
copy of the adenine deaminase gene are used to infect her T cells
- she is still alive and gets a weekly shot of the virus
2000 - X-linked SCID patients are cured when their immune systems are
reconstituted with recombinant viruses carrying the good copy of the gene

91

Gene Therapists Celebrate a Decade of Progress

Science 7 October 2011: vol. 334 no. 6052 29

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Retroviral vector-based gene therapy: problems

2000 - X-linked SCID patients are cured when their immune systems are
reconstituted with recombinant viruses carrying the good copy of the gene

- 5 of 20 patients in France and England develop leukemia

- integration of virus upstream of an oncogene?
- extra copies of the good gene cause cancer?
- trials of this method were halted in 2003 in the US

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Other viral vectors can be used in gene therapy

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Adenoviral vectors can be used in gene therapy

1) the wild type copy of the gene defective in the disease is
inserted into an adenovirus (cold virus) genome
2) the recombinant viruses are exposed to defective cells taken
from the patient
3) the viral genome does not integrate into the host genome
4) these cells can be grown in tissue culture and reintroduced
into the patients body
5) the wild type gene is transcribed and translated
- pro - lack of viral integration reduces risk of insertional mutagenesis
- con - lack of viral integration means constant injections of cells
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Adenoviral vectors can be used in gene therapy

- 1999, Philadelphia
- 18 year old Jessie Gelsinger had a
rare form of liver disease due to
mutation of a gene on the X
chromosome
- he received an injection of
adenovirus containing the wild
type version of the gene and died
4 days later due to a massive
immune response to the virus

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Adenoviral vectors can be used in gene therapy

- 2007 British researchers use

adenoviral gene therapy to treat an
inherited retinal disease caused by
mutations in a single gene
- 28/30 patients had an increase in
vision and no apparent side-effects

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Gene Therapists Celebrate a Decade of Progress

Science 7 October 2011: vol. 334 no. 6052 29

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Newer methods being tested for gene therapy

Other methods to introduce wild type
genes continue to be tested in animal
models
- lipid bilayers
- electroporation
- gene gun bombardment

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Newer methods being tested for gene therapy

- US researchers used viral gene therapy to introduce a red opsin gene
into eyes of adult dichromat squirrel monkey males
- treated males displayed trichromatic vision as shown by behavior tests
- precursor to a treatment for color blindness in humans

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