This chapter discusses various methods for creating transgenic animals, including mice, livestock, birds, and fish. DNA microinjection and embryonic stem cell methods are described for generating transgenic mice and other animals. Applications include disease models, bioreactors to produce human proteins in milk, and increasing disease resistance or growth in livestock and fish. The chapter covers transgenic techniques for mice, cattle, sheep, pigs, chickens, and fish.

1. Chapter 21-Transgenic Animals:
Methodology and Applications
•Transgenic mice: methodology (Retrovirus vector, DNA
microinjection, Engineered embryonic stem cell, Cre-loxP
recombination system, High capacity vectors)
•Transgenic mice: applications (Alzheimer disease, test systems,
conditional regulation, control of cell death)
•Cloning livestock by nuclear transfer
•Transgenic cattle, sheep, goats and pigs
•Transgenic birds
•Transgenic fish

2. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 21
Transgenic Animals
Figure 21.1
Retroviral vectors can be used to create
transgenic animals

3. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 21
Transgenic Animals
Figure 21.3
DNA microinjection is the main
method used to create transgenic
animals

4. Fig. 21.3 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

5. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 21
Transgenic Animals
Figure 21.4
Less than 5% of the
microinjected
fertilized eggs
become transgenic
progeny

6. Creating a transgenic mouse using the
DNA microinjection method
• See
http://bcs.whfreeman.com/lodish7e/#800911__812052__

7. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 21
Transgenic Animals
Figure 21.5
Genetically engineered embryonic
stem (ES) cells can be used to create
transgenic animals, but this method is
labor intensive and used to allow for
gene targeting via homologous
recombination.

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

9. Transgenic animals-Engineered embryonic stem cell
method (used for gene knockouts)
Step 1: Get the ES cells (Fig. 21.5)

10. Step 2: Genetically engineer the ES cells
(Figs. 21.5 and 21.6)

11. Step 3: Place
engineered ES cells
into an early embryo
(Fig. 21.5)
see http://bcs.whfreeman.com/lodish5e/pages/bcs-
main.asp?v=category&s=00020&n=09000&i=09020.01&o=|00510
|00610|00520|00530|00540|00560|00570|00590|00600|0070
0|00710|00010|00020|00030|00040|00050|01000|02000|030
00|04000|05000|06000|07000|08000|09000|10000|11000|12
000|13000|14000|15000|16000|17000|18000|19000|20000|2
1000|22000|23000|99000|&ns=486

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

13. Transgenic mice can be produced with high
capacity vectors
• Generally done by microinjection of numerous genes
contained in a YAC
• Production of mice that can produce human
antibodies is one notable example

14. Transgenic mice/animal: 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 in Fig. 21.19)
• 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)

15. 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_Octobe
r2004.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.

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

17. Fig. 21.22 Cloning
livestock by nuclear
transfer (e.g., sheep)
“Hello Dolly”

18. 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.

19. 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

20. Table 21.2 Some human 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 III (the first transgenic animal drug, an
anticlotting protein, approved by the FDA in 2009)

21. “Enviropigs”
• Transgenic pigs expressing the
phytase gene in their salivary glands
• The phytase gene was introduced via
DNA microinjection and used 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 consequently excrete
large amounts of phosphorus
• “Enviro-pigs” excrete 75% less
phosphorus
• Microinjected an E. coli phytase
gene under the control of a mouse
parotid secretory protein promoter
EnviropigTM an environmentally friendly
breed of pigs that utilizes plant
phosphorus efficiently.

22. Fig. 21.32 Establishing
transgenic chickens by
transfection of isolated
blastoderm cells
• Resistance to viral, bacterial,
and coccidial diseases
• Better feed efficiency
• Lower fat and cholesterol
levels in eggs
• Better meat quality
• Eggs with pharmaceutical
proteins in them

23. 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
• Genetically engineered for a novel pet (Glofish-see http://glofish.com/)

24. Transgenic fish (more detail)
• Salmon were genetically engineered for more rapid growth using the growth
hormone gene under the control of the ocean pout antifreeze protein gene
promoter and 3’ untranslated region (currently under FDA consideration)
• Madaka fish were genetically engineered to serve as biosensors for
environmental pollutants (e.g., estrogens) by using an estrogen-inducible
promoter (the vitellogenin promoter) to control expression of the GFP gene
Fig. 21.33 Fig. 21.34