Chapter 12 – Transgenic Animals

• Transgenic mice: methodology

• Transgenic mice: applications
• Transgenic livestock
• Transgenic poultry
• Transgenic fish
Establishing transgenic mice by
DNA microinjection

• Most commonly used method

• Only 5% or less of the treated eggs
become transgenic progeny
• Need to check mouse pups for DNA
(by PCR or Southerns), RNA (by
northerns or RT-PCR), and protein (by
western or by some specific assay
method)
• Expression will vary in transgenic
offspring: due to position effect and
copy number
Chapter 12
Transgenic Animals

Figure 12.1 Establishing transgenic mice

by DNA microinjection. Note that only 5%
or less of the microinjected fertilized
eggs become transgenic progeny.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
 Creating a transgenic mouse using the
DNA microinjection method
• See https://www.youtube.com/watch?v=ysq-lqp1-Ho
Chapter 12
Transgenic Animals

Figure 12.3 Establishing transgenic mice

with retroviral vectors.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
Chapter 12
Transgenic Animals

Figure 12.6 Establishing transgenic mice

with genetically engineered embryonic
stem cells.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
Transgenic animals-Engineered embyronic
stem cell method (used for gene knockouts)
Step 1: Get the ES cells
Step 2: Genetically engineer the ES cells
 Transgenic
Animals: Using
the Cre-loxP
recombination
system for tissue
or time-specific
gene knockouts
Chapter 12
Transgenic Animals

Figure 12.14 Editing animal genomes using the CRISPR-Cas9 system.

Adapted from Williams et al., 2016, Cold Spring Harb Protoc; doi:10.1101/pdb.top087536.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
Chapter 12
Transgenic Animals
Figure 12.16 RNA interference (RNAi) to knock down expression of a target gene in
transgenic mice. This DNA construct can be introduced into mice embryos by DNA
microinjection or by using a retrovirus (e.g., lentivirus).

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
 Chapter 12
Transgenic Animals

Figure 12.17 Conditional knockdown of

target gene expression using RNA
interference. This strategy takes advantage
of the Cre-loxP recombination system.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
 Transgenic Mice: Applications
• Transgenic mouse models for Alzheimer disease, amyotrophic
lateral sclerosis, Huntington disease, arthritis, muscular dystrophy,
tumorigenesis, hypertension, neurodegenerative disorders,
endocrinological dysfunction, coronary disease, etc.
• Using transgenic mice as test systems (e.g., using RNAi to reduce
levels of proteins [amyloid-b peptide] that contribute to Alzheimer
disease, correcting disease-causing mutations in the dystrophin
gene using CRISPR-Cas9, protection against infectious, such as
pseudorabies virus, by expressing soluble virus receptor proteins)
• Conditional regulation of gene expression (tetracycline-inducible
system)
• Conditional control of cell death (used to model and study organ
failure; involves the organ-specific engineering of a toxin receptor
into the mice and then addition of the toxin to kill that organ)
 Another Transgenic Mouse aApplication:
Marathon Mice
Instead of improving times by fractions of a second,
the genetically enhanced “marathon” mice (above,
on the treadmill in San Diego) ran twice as far and
nearly twice as long as ordinary rodents. The
peroxisome proliferator-activated receptor (PPAR-
delta) gene was overexpressed in these transgenic
mice. For details, see
http://www.salk.edu/otm/Articles/PLoSBiology_Octo
ber2004.pdf
Dr. Ron Evans and one of his genetically
engineered “marathon” mice. The enhanced
PPAR-delta activity not only increased fat
burning, but transformed skeletal muscle fibers,
boosting so-called "slow-twitch" muscle fibers,
which are fatigue resistant, and reducing 'fast-
twitch' fibers, which generate rapid, powerful
contractions but fatigue easily.
 Transgenic Livestock

• Cloning livestock by somatic cell nuclear

transfer
• Pharmaceuticals
• Donor organs
• Disease resistant livestock
• Milk quality
• Animal production traits
• Transgenic poultry
• Transgenic fish
Cloning livestock by
somatic cell nuclear
transfer
(e.g., sheep)-“Hello Dolly”
https://www.youtube.com/watch?v=A
GxdWG6GO3k

Figure 12.29
And now there is pet cloning for a “small” fee…

Nine-week-old "Little Nicky" peers out from August 07, 2008 | Bernann McKinney with one of
her carrying case in Texas. Little Nicky, a  the 5 puppies cloned from Booger, her late pet
cloned cat, was sold to its new owner pit bull. It cost her $50,000. When Booger was
by Genetic Savings and Clone for $50,000 diagnosed with cancer, a grief-stricken McKinney
in December 2004. sought to have him cloned -- first by the now-
defunct Genetic Savings and Clone, and then by
South Korean company RNL Bio.
Chapter 12
Transgenic Animals

Figure 12.29 Cloning livestock by

somatic cell nuclear transfer as depicted
here with sheep. The first cloned sheep
was produced in 1996 and was named
“Dolly”.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
 Transgenic cattle, sheep,
goats, and pigs
• Using the mammary gland as a
bioreactor (see adjacent figure)
• Increase casein content in milk
• Express lactase in milk (to
remove lactose)
• Resistance to bacterial, viral,
and parasitic diseases
• Reduce phosphorous excretion
Chapter 12
Transgenic Animals

Table 12.3

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
 Exogenous proteins expressed in the
mammary glands of transgenic animals
• Erythropoietin
• Factor IX
• Factor VIII
• Fibrinogen
• Growth hormone
• Hemoglobin
• Insulin
• Monoclonal antibodies
• Tissue plasminogen activator (TPA)
• a1-antitrypsin
• Antithrombin (ATyrn)-prevents clotting; 1st approved
recombinant drug produced in an animal (goat);
approved by the FDA in 2009
• See https://www.youtube.com/watch?v=eJCReJbGZKs
Chapter 12
Transgenic Animals

Table 12.2

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
Chapter 12
Transgenic Animals

Figure 12.30 Construct for the expression of human antithrombin in goats’ milk
leading to the production of the first recombinant drug produced in an animal and
approved by the FDA in 2009.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
 “Enviropigs”
• Transgenic pigs expressing the
phytase gene in their salivary
glands
• The phytase gene is introduced
via DNA microinjection and uses
the parotid secretory protein
promoter to specifically drive
expression in the salivary
glands
• Phytate is the predominant
storage form of phosphorus in
plant-based animal feeds (e.g.,
soybean meal)
• Pigs and poultry cannot digest
phytate and thus excrete large
amounts of phosphorus
• “Enviro-pigs” excrete 75% less
phosphorus EnviropigTM an environmentally
• See friendly breed of pigs that utilizes
https://www.youtube.com/watch?v=mAf plant phosphorus efficiently.
CauLF-14
And then there is “transgenic art” with
GFP…
Chapter 12
Transgenic Animals

Figure 12.41 Transgenic chickens

engineered with a small RNA
molecule the inhibits RNA-dependent
RNA polymerase required for
production of new avian influenza
virus.

From Lyall et al., Science. 331:223–226, 2011.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
 Transgenic Fish
• Genes are introduced into fertilized eggs by
DNA microinjection or electroporation
• No need to implant the embryo;
development is external
• Genetically engineered for more rapid
growth using the growth hormone gene
(salmon, trout, catfish, tuna, etc.)
• Genetically engineered for greater disease
resistance
• Genetically engineered to serve as a
biosensor for water pollution
 GloFish: http://www.glofish.com/

Where do GloFish® fluorescent zebra fish come from?

GloFish® fluorescent zebra fish were originally bred to help detect
environmental pollutants. By adding a natural fluorescence gene to the
fish, scientists hope to one day quickly and easily determine when our
waterways are contaminated. The first step in developing these
pollution detecting fish was to create fish that would be fluorescent all
the time. It was only recently that scientists realized the public's
interest in sharing the benefits of this research. We call this the
GloFish® fluorescent fish.
 Transgenic salmon over-expressing GH
• This picture shows the respective
growths of a GM salmon and a
non-GM one at the same age
(Credit: Aqua Bounty).

• The FDA approved this GM

salmon in November 2015!
• https://www.youtube.com/watch?
v=ibkhBTC3Wl4

But why is this GM fish growing so fast?

These GM salmon grow so fast because of a change made to one of the roughly 40,000
genes in their DNA. In normal salmon, the gene that controls the production of growth
hormone (GH) is activated by light, so the fish generally grow only during the sunny
summer months. But by attaching a constitutive "promoter sequence", Aqua Bounty
ended up with salmon that make growth hormone all year round.
Gene construct: Ocean Pout AFP promoter-salmon GH cDNA-3’ Ocean Pout AFP gene
Note AFP=antifreeze protein
 Transgenic Salmon Update: August 2017

The genetically engineered AquaBounty salmon shown here is about twice the size of its
wild kin, although both are roughly the same age. This salmon was approved by the US
FDA in November 2015 and by Canadian authorities in May 2016. The salmon is now
sold in Canada, but not in the US due to political battles, including a provision in the US
government’s budget law for 2017 for labeling this genetically engineered salmon.
Figure 12.42 Construct for the overexpression of growth hormone in Atlantic
salmon used the Chinook salmon growth hormone cDNA under the control of
the promoter and transcription terminator-polyadenylation signals from the
the antifreeze protein (AFP) gene from another fish, the ocean pout.
Figure 12.43 Transgenic medaka fish as biosensors of environmental pollutants.
The vitellogenin gene promoter was used to drive expression of the GFP reporter
gene in medaka fish. Note that the vitellogenin promoter is activated only in the
presence of natural and synthetic estrogenic compounds.
Some Other Genetically Modified Organisms
• See https://www.youtube.com/watch?v=bmi45JLJOgU
• I would encourage you to try to find the
primary research papers on these GMOs if
you have an interest in them in order to get
more and better information on them.