Where do GloFish® fluorescent zebra fish come from?

igocheddarBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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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

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
-
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See also

http://bcs.whfreeman.com/lodish5e/pages/bcs
-
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And for reporter constructs, see
http://bcs.whfreeman.com/lodish5e/pages/bcs
-
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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

Step 2: Genetically engineer the ES cells

Step 3: Place
engineered ES cells
into an early embryo

(Fig. 19.4)

see

http://bcs.whfreeman.com/lodish5e/pages/bcs
-
main.asp?v=category&s=00020&n=09000&i=09020.01&o=|0
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0|03000|04000|05000|06000|07000|08000|09000|10000|110
00|12000|13000|14000|15000|16000|17000|18000|19000|20
000|21000|22000|23000|99000|&ns=486

Transgenic
animals
-
Using
Cre
-
loxP

for
tissue or time
-
specific gene
knockouts

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)

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.

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


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)



a
1
-
antitrypsin


“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

Enviropig
TM

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

And then there is “transgenic art” with
GFP…


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

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.

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.

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.

THE DANGER OF MAMMALIAN CLONING!