Transgenic animal models

Why Transgenic animals are produced?

• Transgenic animals are being produced for various

purposes: For research purposes such as to study the effect of
a drug, its toxicity and side effects and to evaluate the efficacy
of vaccines.

• To produce a better yield of products obtained from animals as

well as their nutritional enrichment such as milk.

• Improving livestock animals was an old age practice for given

genetical traits like milk production, Egg laying efficiency , wool
characteristics through mating and selection. (selective breeding
methods)

• But this traditional method was replaced by nuclear transfer

technique and nuclear cloning techniques by which a single
functional gene or clusters are transferred into chromosomal DNA
of higher organism.
 Breakthrough in molecular biology are happening at an
unprecedented rate.

• One of them is the ability to engineer animals. Transgenic animals, i.e.,

engineered to carry genes from other species, have the potential to improve
human welfare .

• The first genetically modified organism was a bacteria created in 1973 by Stanley
N. Cohen and Herbert Boyer, a bacterium resistant to the antibiotic kanamycin.

• These bacteria contained genetic information from a variety of different species.

• The technology has now produced transgenic animals such as mice, rats, rabbits,
pigs, sheep, and cows.

• The first genetically modified animal, a mouse, was created in 1974 by Rudolf
Jaenisch, and the first plant was produced in 1983.

• Although there are many ethical issues surrounding transgenesis. It has

applications in agriculture, medicine, and industry.
 Transgenic Animal production

• 1980s –beginning era • The idea of introducing gene into egg came
into reality.

• Thus animal whose genetic composition was altered by addition of

exogenous DNA of interest is called transgenic and overall process is
called transgeneisis and transfer of gene is called transgene

• Transgenic animals are those animals that have a foreign gene

deliberately inserted or deleted from their genome.

• The first transgenic animals were mice: In 1974, using a technique

known as nick translation, in which a piece of DNA is radioactively
labeled to allow for its detection, Jaenisch discovered that the DNA of
SV40, following injection into an early-stage mouse embryo, integrated
into all the tissues of the mouse, without causing sarcoma.
 Transgenic animal

Strategy
• Insertion of cloned gene into nucleus of fertilized
egg

• Implementation of such fertilized egg into

receptive female.

• Some of offspring cell may have this newly

introduced gene

• Animal with this gene in the germs cells are

allowed to breed for the establishments of new
genetic lines.

• The production of transgenics provides methods

to rapidly introduce 'new' or modified genes and
DNA sequences into livestock without
crossbreeding or hybridizing. It is a more precise
technique, but not fundamentally different from
genetic selection or crossbreeding in its result.

https://www.sciencedirect.com/science/article/abs/pii/B9780128222652000016
 Transgenic animal
• First transgenic animal was a
Supermouse created by Ralph
Brinster (Univ. Pennsylvania)
and Richard Palmitter (Univ. of
Washington) in 1982.
• It was created by inserting a
human growth hormone gene in
mouse genome.
• The offspring is much larger
than the parents.
• Other animals include pig, goat,
cow, sheep, fish etc.
 Planning a Transgenic
production mouse colony

• Mouse strain - popular

• Colony size
• typical injection 200 embryos (7-10
females s.o.)
• Superovulation efficiency
• Parenting suitability
• Pseudo-pregs
https://www.slideshare.net/damarisb/transgenic-animals-27039475
 (1) Get the nucleotide sequence of the gene of interest. Including upstream
and downstream nucleotides.

agctta cgatc
Gene of Interest
tcgaat gctag
Upstream Downstream
DNA (unique DNA (unique)
to the gene), to the gene,
usually > 1kb usually > 1 kb

(2) Construct the desired DNA sequence (i.e., the transgene), adding
a gene for antibiotic resistance, but keeping the upstream and the
downstream nucleotides.

agctta Desired Gene Antibiotic Resistance Gene

cgatc
tcgaat gctag
 Pronuclear Microinjection

• Microinjection of DNA directly into the pronuclei of fertilized eggs

• Implantation of the microinjected eggs into a surrogate mother
• Allowing the embryos to develop to birth
• Demonstrating that the foreign gene has been stably incorporated
into the host genome and that it is heritable in at least one of the
offspring
• Demonstrating that the gene is expressed and regulated correctly in
the host organism
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
Establishing transgenic mice
with retroviral vectors
(rarely used)
A retroviral vector is produced
by inserting the transgene in place
of part of the viral genome, and a
preparation of infectious viral particles
is produced by introducing the
recombinant virus into tissue culture
cells

A retroviral vector consists of proviral

sequences that can accommodate the gene of
interest, to allow incorporation of both into
the target cells. The vector also contains viral
and cellular gene promoters, such as the CMV
promoter, to enhance expression of the gene of
interest in the target cells.

A transgene is an experimentally constructed piece of

DNA that has integrated into the genome of a
recipient organism. Once integrated into germ cells,
subsequent generations inherit the transgene,
referred to as stable transgene transmission
 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
 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
 Transgenic Cattle

Transgenic bovine cells are selected and fused with

bovine oocytes that have had all of their chromosomes
removed. Once fused with the oocyte, the transgenic cell's
chromosomes are reprogrammed to direct development into
an embryo, which can be implanted into a recipient cow.

Transgenic cows can be used as 'biofactories' to produce

human therapeutic proteins (proteins that are used to treat
diseases). In June 2006, the first therapeutic protein made in
a transgenic animal was approved for use in Europe and the
USA.
Transgenic Cattle
Production
Source: Slide share
Some human proteins expressed in the mammary
glands of transgenic animals
ÒErythropoietin
ÒFactor IX
ÒFactor VIII
ÒFibrinogen
ÒGrowth hormone
ÒHemoglobin
ÒInsulin
ÒMonoclonal antibodies
ÒTissue plasminogen activator (TPA)
Òa1-antitrypsin
ÒAntithrombin III (the first transgenic animal drug, an
anticlotting protein, approved by the FDA in 2009)
 Mouse Transgenesis Methods
pros cons
Pronuclear microinjection
Random integration
Relatively simple and efficient Multicopy insertions
Long transgenes possible ( Strain limitations)
Potentially all species

Lentivral infection
High embryo mortality
Very efficient 9.5 kb packaging limit
Single copy insertions Safety issues (?)
No technical equipment Only random integration
Works in many species

Technically difficult
Long transgenes possible Time consuming
Gene targeting possible Species / Strain limitations
Single copy insertions
S based transgenesis
Trangenic mouse embryo in which the promoter for
a gene expressed in neuronal progenitors
(neurogenin 1)
drives expression of a beta-galactosidase reporter
gene. Neural structures expressing the reporter
transgene are dark blue-green. (Dr. Anne Calof)
 Somatic cell nuclear transfer (SCNT),
Somatic cell nuclear transfer (SCNT), technique in which
the nucleus of a somatic (body) cell is transferred to
the cytoplasm of an enucleated egg (an egg that has had its own
nucleus removed).

Once inside the egg, the somatic nucleus is reprogrammed by

egg cytoplasmic factors to become a zygote (fertilized egg)
nucleus.

The egg is allowed to develop to the blastocyst stage, at which

point a culture of embryonic stem cells (ESCs) can be created
from the inner cell mass of the blastocyst.

Mouse, monkey, and human ESCs have been made using

SCNT; human ESCs have potential applications in both
medicine and research
The most practical application of SCNT is in the reproductive cloning of
farm animals that have exceptional qualities, such as the ability to produce
large quantities of milk.

Reproductive cloning is accomplished by implanting an SCNT-derived

blastocyst into the uterus of a surrogate mother, in which
the embryo develops into a fetus carried to term.

Dolly the sheep, born in 1996, was the first mammal cloned using SCNT.

The technique also could be used to resurrect extinct species; for example,
cells collected from a frozen woolly mammoth could be used as nuclear
donors for enucleated elephant eggs.

Proof of principle for such “resurrection” was provided by an experiment in

which mice were cloned using somatic cell nuclei derived from a mouse that
was frozen for more than 15 years.
 SCNT
Cloning livestock by
nuclear transfer (e.g.,
sheep)
“Hello Dolly”
(12) Problems:
(a) Multiple insertions: too much protein.
(b) Insertion into a life-necessary gene: lethality.
(c) Insertion into a gene leading to gene-silencing: no protein.
(d) Insertion in a different area can lead to differential gene regulation.
(e) Background genotype can be limiting.