A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. Transgenesis is the process by which mixing up of genes takes place. Foreign genes are inserted into the germ line of the animal, so it can be transmitted to the progeny. Transgenic technology has led to the development of fishes, live stock and other animals with altered genetic profiles which are useful to mankind. First transgenic animal was a ‘Supermouse’ created by Ralph Brinster (U Pennsylvania) and Richard Palmiter (University of Washington) in 1982. It was created by inserting a human growth hormone gene in mouse genome. The offspring was much larger than the parents. Mouse – common transgenic expt. Other animals include pig, goat, cow, sheep, fish etc.

1. • BY BAIDEHI MITRA (MPHARM)
• NIRMA UNIVERSITY
TRANSGENIC ANIMAL

2. INTRODUCTION • A transgenic animal is one that carries a foreign gene that has
been deliberately inserted into its genome.
• Transgenesis is the process by which mixing up of genes takes
place.
• Foreign genes are inserted into the germ line of the animal, so it
can be transmitted to the progeny.
• Transgenic technology has led to the development of fishes, live
stock and other animals with altered genetic profiles which are
useful to mankind.
• First transgenic animal was a ‘Supermouse’ created by Ralph
Brinster (U Pennsylvania) and Richard Palmiter (University of
Washington) in 1982.
• It was created by inserting a human growth hormone gene in
mouse genome.
• The offspring was much larger than the parents.
• Mouse – common transgenic expt.
• Other animals include pig, goat, cow, sheep, fish etc.

3. •THE METHODOLOGY
• STEP 1: Construction of a transgene
• Transgene made of 3 parts:
1. Pomoter
2. Gene to be expressed
3. Termination sequence
STEP 2: Introduction of foreign gene into the animal
1. Micro Injection Method
2. Retrovirus mediated gene transfer
3. Embryonic Stem cell - mediated gene transfer
PRODUCTION OF TRANSGENIC ANIMAL

4. MICROINJECTION METHOD
• This procedure involves delivery of foreign gene into
the living cell by using fine glass micro pipettes and
micro injection.
• A female animal is super-ovulated and eggs are
collected.
• The eggs are fertilized in vitro.
• The transgene containing solution is injected into the
male pronuclear using a micropipette.
• Eggs with the transgenes are kept overnight in an
incubator to develop to a 2 cell stage.
• The eggs are then implanted into the uterus of a
pseudo pregnant female (female which has been
mated with a vasectomized male the previous night).

5. EMBRYONIC STEM CELL METHOD
• Transgenic animals can be created by manipulating
embryonic stem cells.
• ES cells are obtained from the inner cell mass of a blastocyst.
• Transgene is incorporated into the ES cell by
1. Microinjection
2. By a retro virus
3. By electroporation
• Transgenic stem cells are grown in vitro.
• Then they are inserted into a blastocyst and implanted into a
host's uterus to grow normally.

6. BLASTOCYST MICRO INJECTION

7. • Step 3: Screening for transgenic positives
A. Transgenic progenies are screened by PCR to examine the site of
incorporation of the gene
B. Some transgenes may not be expressed if integrated into a transcriptionally
inactive site.
• Step 4: Further animal breeding is done to obtain maximal expression.
A. Heterozygous offspring's are mated to form homozygous strains.

8. • A retrovirus is a virus that carries its genetic material in the form of RNA rather than
DNA.
• This method involves:
• 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.
Retrovirus-Mediated Gene Transfer

9. • Inserted gene has multiple functions.
• Breeding problems.
• Sometimes leads to mutagenesis and functional disorders.
• Low survival rate of transgenic animals.
• Very expensive
PROBLEMS

10. SOME EXAMPLES OF TRANSGENIC ANIMALS

11. Transgenic fish
• SUPER FISH
1. Increased growth and size Growth hormone gene
inserted. into fertilized egg.
2. Transgenic salmon grows about 10 - 11 times
faster than normal fish.
• GLO FISH
1. GM freshwater zebra fish (Danio rerio)
2. Produce by integrating a fluorescent protein gene
from jelly fish into embryo of fish.

12. TRANSGENIC MOUSE • Alzheimer mouse
A. In the brain of Alzheimer's patients, dead nerve
cells are entangled in a protein called amyloid.
B. Mouse made by introducing amyloid precursor
gene into fertilized egg of mice.
• ONCOMOUSE
1. Mouse model to study cancer (nude mice)
(glowing mice).
2. Made by inserting activated oncogenes.
3. p53 Null (p53-/-) mice have an average time to
tumor development of 4.5 months, while half
of the heterozygous (p53+/-) mice develop
tumors by 18 months. The p53-deficient mice
have been particularly valuable in examining
the effects of p53 loss on tumor progression.
In addition, the mice hold significant promise
as tools to assess carcinogens, teratogens,
chemo preventative agents, and cancer
therapeutic regimens.
1ST AIDS MOUSE – SCID MOUSE
(LACKED IMMUNE SYSTEM)
GENETICALLY ENGINEERED SKIN CELLS
GRAFTED ONTO MICE CAN TREAT THE
ANIMALS’ DIABETES AND OBESITY

13. What is P53 gene?
• A gene that makes a protein that is
found inside the nucleus of
cells and plays a key role in
controlling cell division and cell death.
Mutations (changes) in the p53 gene
may cause cancer cells to grow and
spread in the body.

14. Transgenic Animals For Diabetes and other Disease

15. Sr. No. Model Category Obese type 2 diabetic model Non-obese type 2 diabetic model
1 Spontaneous or genetically derived diabetic animals ob/ob mouse
db/db mouse
KK mouse
KK/Ay mouse
NZO mouse
NONcNZO10 mouse
TSOD mouse
M16 mouse
Zucker fatty rat
ZDF rat
SHR/N-cp rat
JCR/LA-cp rat
OLETF rat
Obese rhesus monkey
Cohen diabetic rat
GK rat
Torri rat Non obese C57BL/6KK/Ay mouse(Akita)
mutant mouse
ALS/Lt mouse
2 Transgenic/knock-out
diabetic animals
Beta 3 receptor knockout mouse
Uncoupling protein (UCP1)
knock-out mouse
Transgenic or knock out mice involving
genes of insulin and insulin receptor
and its components of downstream
insulin signaling e.g. IRS-1, IRS-2,GLUT-4, PTP-1B
and othersPPAR-gama tissue specific
knockoutmouseGlucokinase or GLUT 2 gene
knockoutmiceHuman islet amyloid
polypeptideoverexpressed rat (HIP rat)
Classification of type 2 diabetes in animals

16. Obesity:
• Obesity is characterized by an excessive development of fat mass, which is a consequence of
increased size of adipocytes and/or increased number of adipocytes and may have and adverse
effect on health, leading to reduced life expectancy and /or increased health.
• Genetic models of obesity include
1. db/db mice,
2. ob/ob mice,
3. Zucker diabetic fatty rats and
4. Otsuka Long-Evans Tokushima Fatty rats.
• These models are useful in evaluating specific molecular mechanisms that may be involved in
development of obesity in rodents.
• As an example, several of these models have mutations in the leptin gene or receptor.

17. ob/ob (C57BL/6J-ob/ob) Mice
• These mice inherited a monogenetic autosomal recessive mutation in the leptin gene on
chromosome 6 and developed obesity after 4 weeks of age.
• They showed an increased body weight compared to their lean littermates at all ages.
• The presence of impaired glucose tolerance was found after 12 weeks of age.
• These mice developed left ventricular hypertrophy with decreased cardiac function at 24
weeks of age, cardiac fibrosis after 20 weeks of age and hepatic steatosis and inflammation
at 12 weeks of age.

18. db/db (C57BL/KsJ-db/db) Mice
• These mice have inherited an autosomal recessive mutation in the leptin receptor gene
present on chromosome 4 leading to higher body weights than their lean littermates after 6
weeks of age.
• Fasting blood glucose concentrations were higher after 8 weeks of age and these mice
showed increased plasma concentrations of triglycerides, total cholesterol and
nonesterified fatty acids along with reduced HDL/LDL cholesterol ratio after 13 weeks of
age.
• Hyperinsulinaemia and impaired glucose tolerance were observed after 12 weeks of age.

19. Zucker Diabetic Fatty Rats (fa/fa)
• Diabetic Zucker fatty rats (ZDF), a model of early onset obesity, have a mutation in the
leptin receptor gene.
• Serum cholesterol concentrations were slightly increased in ZDF rats compared to lean
Zucker rats at 10 weeks of age whereas the serum concentration of cholesterol was ~2.5
times higher compared to lean Zucker rats at 20 weeks of age.
• These rats also developed endothelial dysfunction after 12 weeks of age.
• Increased hepatic triglyceride deposition was observed after 20 weeks of age in ZDF rats.
• ZDF rats also showed increased serum markers of inflammation such as TNF-α and IL-1β
after 26 weeks of age.
• Albuminuria was present at the age of 31 weeks with thickening of basal membrane and
glomerular fibrosis after 47 weeks.

20. Otsuka Long-Evans Tokushima Fatty Rats
• Otsuka Long-Evans Tokushima Fatty (OLETF) rats have been used as a rat model of
human diabetes and obesity.
• Pancreatic acini cells in OLETF rats were insensitive to the actions of cholecystokinin
(CCK), which controls food intake, due to the absence of CCK-1 receptors.
• Due to the lack of CCK-1 receptors, the average meal size and overall food intake were
higher in OLETF rats.
• Male and female OLETF rats were similar in body weight to lean Long-Evans Tokushima
rats at the time of weaning but they became 30–40% heavier than age-matched lean Long-
Evans Tokushima Otsuka rats after 20 weeks.

21. Transgenic Animal Model Used In Screening
Of Anti-Hypertensive Agent

22. Genetic Hypertension
• In 1963, Okamoto and Aoki introduced a new model of experimental hypertension that
required no physiological pharmacological or surgical intervention. The so called
spontaneous hypertensive rat (SHR).
• Spontaneously hypertensive rats (SHRs) were originally inbread from Wistar rats and their
Wistar-Kyoto inbred non-hypertensive controls.
• These Rats develop hypertension at above 4-6 weeks of age without physiological,
pharmacological or surgical intervention however, environmental factors affect the
development of hypertension.

23. Transgenic hypertension models
• Transgenic hypertension models can be generated by over expression of a specific gene.
This is an excellent model study the role of a specific gene in the pathogenesis of
hypertension.
• A representative of this type of hypertension is the TGR(mREN2)27 transgenic rat
developed by Mullins et al.
• Although the model is not representative of human hypertension, it does allow in vivo
analysis of the consequence of severe, monogenic activation of the hypertensive damage
that can be expected from an activated RAAS.

24. Medicine
• Xenotransplantation
• Patients die every year for lack of a replacement heart, liver, or kidney. Transgenic pigs may
provide 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 liter.
• 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
APPLICATION

25. 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.

26. 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 and
weaving them into thread, the
scientists can create a light, tough,
flexible material that could be used in
such applications as military uniforms,
medical micro sutures, 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.

27. GOAT WITH GENE OF SPIDER SILK

28. Thank
you