Chapter 21 Transgenic Animals Figure 21.5

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Dec 12, 2012 (4 years and 9 months ago)

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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

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

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

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

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

Creating a transgenic mouse using the

DNA microinjection method


See
http://bcs.whfreeman.com/lodish7e/#800911__812052__


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.

Establishing
transgenic animals
using engineered
embryonic stem
(ES) cells

But what are ES
cells?

Transgenic animals
-
Engineered embryonic stem cell
method (used for gene knockouts)

Step 1: Get the ES cells (Fig. 21.5)

Step 2: Genetically engineer the ES cells

(Figs. 21.5 and 21.6)

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

Transgenic
animals
-
Using Cre
-
loxP

for tissue or
time
-
specific gene
knockouts

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


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)

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.

And then there is

transgenic art


with GFP…


Fig. 21.22 Cloning
livestock by nuclear
transfer (e.g., sheep)


Hello Dolly


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.

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


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)


a
1
-
antitrypsin


Antithrombin III (
the first transgenic animal drug, an
anticlotting protein, approved by the FDA in 2009
)




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

Enviropig
TM

an environmentally friendly
breed of pigs that utilizes plant
phosphorus efficiently.

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

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/
)



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