Biotechnology and Genetic Engineering-PBIO 450/550

roachavocadoBiotechnology

Dec 14, 2012 (4 years and 6 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

Fig. 21.1 Establishing
transgenic mice with
retroviral vectors

(rarely used)

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

Creating a transgenic mouse using the

DNA microinjection method


See

http://bcs.whfreeman.com/lodish5e/pages/bcs
-
main.asp?v=category&s=00020&n=09000&i=09020.02&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|0
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000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=538



See also

http://bcs.whfreeman.com/lodish5e/pages/bcs
-
main.asp?v=category&s=00010&n=09000&i=09010.10&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|0
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000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=661



And for reporter constructs, see
http://bcs.whfreeman.com/lodish5e/pages/bcs
-
main.asp?v=category&s=00010&n=15000&i=15010.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|0
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000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1322

Establishing
transgenic animals
using engineered
embryonic stem
(ES) cells

But what are ES
cells?

Transgenic animals
-
Engineered
embyronic

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