Transgenic animals are created using two main methods: DNA microinjection and manipulating embryonic stem cells. Examples of transgenic animals include GH-mice with increased growth, marathon mice with enhanced endurance, and goats producing the human blood clotting protein Antithrombin in their milk. Transgenic fish like GloFish have been genetically engineered to fluoresce for environmental monitoring. Mammalian cloning is done through nuclear transfer as demonstrated by Dolly the sheep. Transgenic technologies allow for applications like disease modeling and industrial production of human therapeutic proteins from the milk of livestock.

1. Animal Genetic Engineering:
Methodology and Applications
• Transgenic animals created by two methods
1.DNA microinjection
2.Embryonic stem cells & gene knock-outs/ also Cre-loxP
• Transgenic animals: some examples
 GH-mice
 Marathon mice
 ATyrn goats
• Transgenic fish: some examples
 GloFish (GFP-zebra fish)
 GH-salmon
• Mammalian Cloning by nuclear transfer
 Dolly the sheep
 Pet cloning

2. 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

3. Creating a transgenic mouse using the
DNA microinjection method
• See https://www.youtube.com/watch?v=HiF9RDZJ0Pg
• See https://www.youtube.com/watch?v=ysq-lqp1-Ho

5. Establishing
transgenic
animals using
engineered
embryonic stem
(ES) cells
But what are ES
cells?

6. Transgenic animals-Engineered embyronic
stem cell method (used for gene knockouts)
Step 1: Get the ES cells

7. Step 2: Genetically engineer the ES cells

8. Step 3: Place
engineered ES cells
into an early embryo
(Fig. 19.4)
see the Chapter 5 video

9. Transgenic
animals-Using
Cre-loxP for
tissue or time-
specific gene
knockouts

10. Transgenic mice: applications
• Transgenic 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., protein [CFTR]
secretion into milk, protection against mastitis caused by
Staphylococcus aureus using a modified lysostaphin gene)
• 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)

11. Another Transgenic mouse application:
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.

12. 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

13. 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

14. “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
• See
https://www.youtube.com/watch?v=mAf
CauLF-14
EnviropigTM an environmentally
friendly breed of pigs that utilizes
plant phosphorus efficiently.

15. And then there is “transgenic art” with
GFP…

16. 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

17. 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.

18. Transgenic salmon over-expressing GH
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
• 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

19. Cloning livestock by
nuclear transfer
(e.g., sheep)-“Hello Dolly”
https://www.youtube.com/watch?v=AG
xdWG6GO3k

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