Chapter 7/8: Animal Genetic Engineering Methodology and Applications

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Chapter 7/8 Animal Genetic Engineering
Methodology and Applications

• Transgenic animals created by 2 methods
• 1) Microinjection of fertilized eggs
• 2) Transfection and implantation of embryonic
  stem cells
• .(gene knock-outs Cre-loxP recombination
  system)
• Transgenic animals some examples
• Transgenic fish some examples
• Mammalian Cloning by nuclear transfer

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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

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Creating a transgenic mouse using theDNA
microinjection method

• See http//bcs.whfreeman.com/lodish5e/pages/bcs-ma
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• And for reporter constructs, see
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Establishing transgenic animals using engineered
embryonic stem (ES) cellsBut what are ES cells?
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Transgenic animals-Engineered embyronic stem cell
method (used for gene knockouts)Step 1 Get the
ES cells
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Step 2 Genetically engineer the ES cells
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Step 3 Place engineered ES cells into an early
embryo(Fig. 19.4)see http//bcs.whfreeman.com/lo
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Transgenic animals-Using Cre-loxP for tissue or
time-specific gene knockouts
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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)

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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_Octob
er2004.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.
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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

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Some exogenous proteins that have been 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

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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

EnviropigTM an environmentally friendly breed of
pigs that utilizes plant phosphorus efficiently.
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And then there is transgenic art with GFP
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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

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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.
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Transgenic salmon over-expressing GH
Above is a picture showing the respective growths
of a GM salmon and a non-GM one at the same age
(Credit Aqua Bounty). 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 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.
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Cloning livestock by nuclear transfer (e.g.,
sheep)Hello Dolly
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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.
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THE DANGER OF MAMMALIAN CLONING!