2. What are Transgenic Animals?
• An animal that gains new genetic
information from the addition of
foreign DNA is described as
Transgenic while the introduced
DNA is called the transgene.
• From the early 1980s, fruit flies,
fish, sea urchins, frogs, laboratory
mice and farm animals, such as
cows, pigs, and sheep have been
successfully produced.
3. History :
• In 1974 Rudolf Jaenisch created a transgenic mouse by introducing foreign
DNA into its embryo, making it the world’s first transgenic animal. However it
took another eight years before transgenic mice were developed that passed
the transgene to their offspring.
• Genetically modified mice were created in 1984 that carried
cloned oncogenes, predisposing them to developing cancer.
• Mice with genes knocked out (knockout mouse) were created in 1989. The
first transgenic livestock were produced in 1985 and the first animal to
synthesise transgenic proteins in their milk were mice, engineered to produce
human tissue plasminogen activator in 1987.
• The first genetically modified animal to be commercialised was the GloFish,
a Zebra fish with a fluorescent gene added that allows it to glow in the dark
under ultraviolet light.
• The first genetically modified animal to be approved for food use
was AquAdvantage salmon in 2015. The salmon were transformed with
a growth hormone-regulating gene from a Pacific Chinook salmon and a
promoter from an ocean pout enabling it to grow year-round instead of only
during spring and summer.
4. Transgenesis Methodology
For transgenesis, DNA can be introduced into animal by
one of the following methods;
• Retroviral meditated transgenics
• Microinjection
• Introduction of genetically engineered embryonic
stem cells into an early stage developing embryo
prior to implantation into a receptive female.
• Sperm meditated transfer
• Gene Gun
5. Retroviral Vectors
• Infect early stage embryo with replication-
defective retrovirus.
• A retrovirus is a virus that carries its genetic
material in the form of RNA rather than
DNA.
• Retroviruses used as vectors to transfer
genetic material into the host cell, resulting
in a Chimera, an organism consisting of
tissues or parts of diverse genetic
constitution .
• Chimeras are inbred for as many as 20
generations until homozygous (carrying the
desired transgene in every cell) transgenic
offspring are born
• The method was successfully used in 1974
when a simian virus was inserted into mice
embryos, resulting in mice carrying this DNA.
6. Common Vectors Used:
Vector Origin Insert size range
• Lambda vectors Bacteriophage λ up to 30 kb
• Cosmid Bacteriophage λ up to 40 kb
• P1 artificial chrom Bacteriophage P1 80-90 kb
• Bacterial artificial chrom. Large Bacteria plasmid 100-300 kb (F factor)
• Yeast chrom. (YAC) Yeast chromosome 100-1000 + kb
+ means indefinite.
7.
8. Pronuclear Microinjection
• The mouse was the first animal to undergo
successful gene transfer using DNA
microinjection.
• This method involves:
• Transfer of a desired gene construct (of a
single gene or a combination of genes that are
recombined and then cloned) from another
member of the same species or from a different
species into the pronucleus of a reproductive
cell.
• The manipulated cell, which first must be
cultured in vitro (in a lab, not in a live animal)
todevelop to a specific embryonic phase, is then
transferred to the recipient female.
9. The eggs are inoculated immediately
with the transgene, briefly;
– embryo at the pronuclear stage is
held in place by suction.
– a micro needle loaded with a
suspension of plasmid DNA will be
prepared.
– It is introduced through the zona
pellucida and plasma membrane
into the most accessible
pronucleus (usually the male) and
several hundred molecules of the
recombinant DNA are injected in a
volume of approximately 1
picoliter (p1).
– on a good day several hundred
eggs can be injected.
10. Engineered Embryonic Stem Cell
Method:
• In this method, cells from the Inner Cell
Mass (ICM) of early embryos blastocysts (a
stage of a developing mouse embryo) will
be used.
• These cells can be grown in cell culture and
still retain the capability of differentiating
into other cell types including germ line
cells after they are introduced into another
blastocyst embryo.
• Such cells are called pluripotent (multi)
embryonic stem (ES) cells. These cells can
be easily manipulated by genetic
engineering without changing their
pluripotency
11.
12. Sperm Meditated Gene Transfer
• Sperm-mediated gene transfer (SMGT)
is a transgenic technique that
transfers genes based on the ability
of sperm cells to spontaneously bind to
and internalize exogenous DNAand
transport it into an oocyte during
fertilization to produce genetically
modified animals.
• Uses “linker proteins” to attach DNA to
sperm cells
13. Chemical Methods:
• Calcium phosphate method; involves the formation of a fine
DNA/calcium phosphate co-precipitate which first settles on
the cells and then internalized by endocytosis.
• The precipitate must be formed freshly at the time of
transfection. The DNA escapes and reaches the nucleus and
can be then expressed. Since the cells must be coated by the
calcium complex, monolayers of cells must be used for
maximum efficiency. However, this method gives only 1-2%
transfection efficiency.
14. Electroporation:
• It is a physical transfection technique
involves creating transient nano-meter size
pores in the cell membrane by exposing
cells to a brief pulse of electricity. The most
critical parameter is the intensity and
duration of electrical pulse.
• Electroporation can be used for in vivo gene
transfer particularly for surface or near
surface tissue such as skin, muscle and
certain tumors or even internal tissues such
as liver. This can be achieved by direct
application of electrodes to the skin
following shaving and mild abrasion with
the DNA being injected into the skin before
electroporation.
15. Application of Transgenic
Animals
Transgenic Mice as Animal Models of Human Diseases
Animal models for human illnesses are useful for studying the pathogenesis of
diseases as well as for developing and testing new therapies. Human diseases
can be induced in transgenic mice by expression of transferred genes, or by
insertional disruption of endogenous sequences.
Some examples of models created by transgene expression are listed below.
• Hepatitis B is a human disease that lacks a readily workable animal model.
Introduction of the HBSAg gene into mice results in transgenic mice that mimic
the carrier state with production of HBsAg in the liver but with an absence of
disease.
Transgenic Mice as Models for Gene Therapy
• Genes can be inserted into transgenic animals and function to alleviate disease
states, such model systems can be of great importance in improving our
understanding of the potential for gene transfer as an approach to treatment of
diseases.
• Mice with growth hormone deficiency are markedly reduced in size and males
suffer from infertility. Introduction of the growth-hormone gene into these
animals leads to growth which exceeds that of normal animals and restores male
fertility.
16. Goals of producing transgenic cattle
• To change the constituents of milk. For example the amount of cheese
produced from milk is directly proportional to the amount of k-casein content
of the milk so if a transgene is constructed to produce milk with higher
amounts of k-casein, then the production of cheese will increase
proportionally.
• Production of transgenic cows with modified genes to produce lactose free
milk could solve the problem of those who have lactose intolerance.
• For livestock in general, attempts to produce animals with inherited resistance
to bacterial, viral, and parasitic disease is a goal. Example of major diseases
that affect the livestock are mastitis in cows, neonatal dysentery in swine, fowl
cholera.
• If the basis of each of these is a single gene that will be responsible for the
resistance, then it might be possible to produce transgenic animals that carry
this gene.
17. Transgenic Sheep
• Transgenesis research with sheep, goat or pigs has
concentrated in the most part on utilizing their
mammary glands as bioreactors for production of
pharmaceutical proteins.
Example; Production of transgenic sheep that
produces anti-trypsin in their milk; This protein is
a potential treatment for cystic fibrosis.
• What can we use transgenic chicken for?
• To improve the genetic makeup of the existing
strains with respect to
– resistance to avian viral and coccidial diseases,
– better feed efficiency,
– lower fat and cholesterol in eggs and
– better meat quality.
• The egg with its high protein content could be
used as a source of pharmaceutical proteins
18. Medicine
• Xenotransplantation
Patients die every year for lack of a replacement heart,
liver, or kidney. Transgenic pigs mayprovide the transplant
organs needed to alleviate the shortfall.
• Nutritional supplements and pharmaceuticals
Products such as insulin, growth hormone, and blood anti-
clotting factors may soon be or have already been
obtained from the milk of transgenic cows, sheep, or
goats. In 1997, the first transgenic cow, Rosie, produced
human protein-enriched milk at 2.4 grams per litre.
• Human gene therapy
Human gene therapy involves adding a normal copy of a
gene (transgene) to the genome of a person carrying
defective copies of the gene. The potential for treatments
for the 5,000 named genetic diseases is huge and
transgenic animals could play a role.
19. Agriculture
• Breeding
Farmers have always used selective breeding to
produce animals that exhibit desired traits
(e.g.,increased milk production, high growth
rate).Transgenesis made it possible to develop traits in
animals in a shorter time and with more precision. In
addition, it offers the farmer an easy way to increase
yields.
• Quality
Transgenic cows exist that produce more milk or milk
with less lactose or cholesterol, pigs and cattle that
have more meat on them, and sheep that grow more
wool.
• Disease resistance
Scientists are attempting to produce disease-resistant
animals, such as influenza-resistant pigs,but a very
limited number of genes are currently known to be
responsible for resistance to diseases in farm animals
20. Industries
• In 2001, two scientists at Nexia Biotechnologies
in Canada spliced spider genes into the cells of
lactating goats. The goats began to manufacture
silk along with their milk and secrete tiny silk
strands from their body by the bucketful. By
extracting polymer strands from the milk
andweaving them into thread, the scientists can
create a light, tough, flexible material that could
beused in such applications as military uniforms,
medical microsutures, and tennis racket strings.
• Toxicity-sensitive transgenic animals have been
produced for chemical safety testing.
• Microorganisms have been engineered to
produce a wide variety of proteins, which in turn
can produce enzymes that can speed up
industrial chemical reactions.
• Biopharming
21. Advantages and Disadvantages
Advantages
• Increased growth rate
• Improved disease resistance.
• Improved food conversion rate.
• Increased muscle mass.
• Improved nutritional value.
• Improved wool quality
Disadvantages
• Inserted gene has multiple
functions.
• Breeding problems.
• Sometimes leads to mutagenesis
and functional disorder.
• Low survival rate of transgenic
animals.
