TRANSGENIC ANIMALS METHOD OF DEVELOPMENT APPLICATIONS

1. TRANSGENIC ANIMALS
PRESENTED BY
NIHARA ANILKUMAR
1ST YEAR M.PHARM
PHARMACOLOGY

2. CONTENTS:
• INTRODUCTION
• DEFENITION
• GOALS
• PURPOSE
• BENEFITS AND RISKS
• TYPES OF TRANSGENIC ANIMALS
• METHOD OF PRODUCTION
• MAINTANENCE
• APPLICATIONS

3. INTRODUCTION
• Organisms containing integrated sequences of cloned
DNA , transferred using techniques of genetic
engineering are called transgenic animals.
• Genetically modified animals are proving ever more
vital in the development of new treatments and cures
for many serious diseases.
• Transgenesis is a radically new technology for altering
the characteristics of animals by introducing the
foreign genetic material.

4. Definition:
• An organism that is engineered to carry a foreign gene, or
transgene of choice as part of its own genetic material.
• These are genetically modified organisms with DNA
from another source inserted into their genome.
• A large number of transgenic animals have been created
such as Mice, Cows, Pigs, Sheep, Goats, Fish, and Frogs.

5. • The first transgenic animal was Rudolf Jaenisch’s mice.
• Jaenisch, a professor of biology, in 1970 successfully
introduced an outside gene into the mice embryos. The
mice and their offspring developed leukemia.
E.g., transgenic cattle were created to produce milk
containing particular human proteins, which may help in
the treatment of human emphysema.

6. • Research into animal and human disease
• Improve livestock animals
• Use of animals as bioreactors
GOALS :

7. Purpose:
• These animals are very useful for delineating the function
of newly discovered genes as well as for producing useful
proteins in large animals.
• Transgenic animals are useful as disease models and
producers of substances for human welfare.
• These animals are genetically manipulated to exhibit disease
symptoms so that effective treatment can be studied.
e.g. - The OncoMouse (Harvard mouse), carrying a gene that
promotes the development of various human cancers.

8. BENEFITS AND RISKS
BENEFITS:
• Desired characteristic
may be introduced for
animal that require few
feed supplements as well
as medical treatments.
• A desired characteristic
of offspring could be
established in one
generation.
• .The characteristic
required can be chosen
with greater specificity
and accuracy.
RISKS:
• Insertion of foreign gene
may upset the expression
of the genome.
• Normal reproduction
may result in a transgene
being released to the wild
population.

9. TYPES OF TRANSGENIC
ANIMALS
1. TRANSGENIC COW
• Transgenic cows carrying extra copies
of two types of casein genes produce
13% more milk protein.
• Currently the milk from these animals is
under FDA review.
:
• They are used in organ transplant
harvesting and study of human membrane co-
factor protein.
• Breeding problem and Mutation will occur.
2. ENVIRO PIG:

10. 3.TRANSGENIC FISH
 Tilapia
 Salmon/trout
 Catfish
These can grow up to 6 times faster than wildtype fish and most have
extracopies of growth hormone (GH) gene.

11. 4.TRANSGENIC SHEEP
Tracy is the first transgenic animal to
produce a recombinant protein in
her milk.
uses of transgenic sheep:
It is used as a model for studying;
 Human blood clotting factor viii
 Transplantation
 Haematology
 Biological product manufacturing
 Recombinant DNA
 Drug production in milk
Disadvantages
o Difficult procedure
o Failed in vitro fertilization
o Expensive

12. 5.Transgenic Monkey:
Its so similar to human hence it used in clinical
trail used for studying :
• HIV
• Huntington’s disease
Disadvantages
o Expensive
o Difficulty in Breeding problem

13. 6.TRANSGENIC MICE
ADVANTAGES
 Gene mutation
 Alzheimer's disease
 Hypertension
 Atherosclerosis
 Cardiac hypertrophy
 Human leukocyte antigen
 Human gastric carcinoma
 Making poliovirus vaccine
 Different type of cancer
 HIV studies
 Ocular inflammation
DISADVANTAGES:
– Expensive
– Gene can only be added ,not deleted
– Embryos are not easily accessible
for manipulation

14. 7.TRANSGENIC RABBIT:
Advantages
• Gene mutation
• Cell metabolisms
• Hemorrhagic disease
• Eye disease
• Heart problems
• Atheroschlerosis

15. KNOCK OUT MOUSE
• A knockout mouse or knock-out mouse is a genetically
modified mouse (Mus musculus) in which researchers
have inactivated, or "knocked out", an existing gene by
replacing it or disrupting it with an artificial piece
of DNA.
• They are important animal models for studying the role of
genes which have been sequenced but whose functions
have not been determined.
• By causing a specific gene to be inactive in the mouse, and
observing any differences from normal behaviour or
physiology, researchers can infer its probable function.

16. • The target gene disruption can be carried out by
incorperating a DNA sequence, usually a selectable
marker gene into the coding region.
APPLICATIONS OF GENE KNOCK OUT
 Biochemical and pathological basis of several
human diseases can be approximately understood by
inactivating specific genes.
 About 200 knock out mouse have been created to
serve as animal models for study of large number of
disorders.

18. METHODS TO PRODUCE
TRANSGENIC ANIMALS
• EMBRYONIC STEM CELL METHOD.
• PRONUCLEUS METHOD.
• RETROVIRUS MEDIATED GENE TRANSFER.
• SPERM MEDIATED GENE TRANSFER.

19. EMBRYONIC STEM-
CELL METHOD
This method involves:
 Isolation of totipotent stem cells (stem cells that can
develop into any type of specialized cell) from embryos.
 The desired gene is inserted into these cells.
 Cells containing the desired DNA are incorporated into the
host's embryo.

20. STEPS :
Embryonic stem cells
Formation of gametocytes
Injection into blastocyst
Injection into foster mother
Formation of new individual

21. PRONUCLEUS METHOD /
(DNA MICROINJECTION)

22. • DNA microinjection or pronucleus method is the most common
method used to create transgenic animals.
• The first step of this process is to expose fertilized eggs to
transgene before cell differentiation so that the gene will be
present in the animal while it evolves.
• The egg and sperm must then be fertilized in vitro before
the pronuclei fuse, and the pronucleus is microinjected with
the recombinant DNA that includes the transgene.
• After injection fertilized eggs are then placed into the oviducts
of a foster mother, who is able to carry the eggs after mating
with a vasectomised male.
• When the offspring are born, a tissue sample is extracted and
screened for presence of the transgene.

23. STEPS:
Formation of DNA as in the first method
.
Fusion of male pronucleus with desired DNA.
Formation of diploid zygote
Mitotic division
Formation of two-cell embryo
.
Transferring to foster mother.
Formation of new individual

25. RETROVIRUS MEDIATED
GENE TRANSFER
 The word “retro” means, when the virus vectors infect a host
cell, the viral RNA is reverse transcribed in the cytoplasm
making linear double-stranded DNA.
 A retrovirus is a virus that carries its genetic material in the
form of RNA rather than DNA.
 In this method, retroviruses are 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.
 Mice embryo is infected with a retrovirus which carry the
new gene.
 When cells are infected by retroviruses, the resultant viral
DNA, after reverse transcription and integration, becomes a
part of the host cell genome to be maintained for the life of
the host cell.

27. Sperm-mediated gene
transfer (SMGT)
• In 1989 Lavitrano et al. described a simple and efficient
technique, sperm-mediated gene transfer to produce
transgenic mice.
• Use of “Linker protein" to attach DNA to sperm which
transfer the new DNA during fertilization.
• In this technique, DNA was mixed with sperm cells before
in vitro. 30% of offspring mouse were integrated foreign
DNA.
• The basic principle of sperm-mediated gene transfer is:
seminal plasma-free sperm cells are suspended in the
appropriate medium, and then incubated with DNA.
• The resultant DNA-carrying sperms are then used to
fertilize eggs, via in vitro fertilization or artificial
insemination or, in the case of aquatic animals, via
waterborne (natural) fertilization.

29. • Establishing and Maintaining Lines Requires
Aggressive Breeding
 When transgenic founders are first identified, it is important to
breed them aggressively until the line is established.
 This is because the presence and expression of a transgene can
compromise fertility and because the founder may be a mosaic,
with the transgene present in only a subset of germ cells.
 For these reasons, lab animal care personnel should be
prepared to provide the necessary equipment and space.
MAINTENENCE

30. • Embryo Freezing
 Embryo freezing is the process of preserving an embryo at sub-
zero temperatures, generally at an embryogenesis stage
corresponding to pre-implantation, that is from fertilisation to
the blastocyst stage.
 When freezing embryos for transgenic strain preservation, it is
important to realize that large numbers of embryos are required
to assure rapid reconstitution of the strain.
 Embryo freezing also requires a backup system to secure
against freezer breakdown, and the availability of skilled labor.
 Establishment of a freezing program, therefore, requires
significant cost, an adequate organizational infrastructure, and
time for strain reconstitution.

31. • COST CONTAINMENT
 When the sequence to be microinjected is poorly designed,
mice are produced that give less than the expected result.
 This situation frequently leads to new rounds of
microinjection with new constructs, but few investigators
discard the previous mice.
 Therefore, because the effort to produce transgenic mice is
substantial and because animal care costs are high, it is
advisable to plan experiments carefully.
 When several lines are produced with a single construct, it
may be advisable to discontinue some of the lines.

32. • REPRODUCTIVE MANAGEMENT
 Since male mice can be maintained for a significant
period of time without losing fertility, it is possible to
maintain some lines by simply keeping 1-2 males on
the shelf for up to 6 months without breeding them,
provided that the males are known to transmit the
transgene effectively.

33. APPLICATIONS
• Transgenic technology holds a great potential in
different-different groups on the basis of benefits
of these animals to human welfare can be
grouped into areas:
1. Agricultural
2. Medicinal
3. Industrial field

34. AGRICULTURE
(A) Breeding
 Farmers have always used selective breeding to produce
animals that exhibit desired traits (e.g., increased milk
production, high growth rate).
 Traditional breeding is a time-consuming, difficult task.
 Researchers have now used gene transfer to improve the
productivity of livestock which offers farmers an easy way
to increase yields.
 Scientists can improve the size of livestock genetically.
 Transgenic cows exist that produce more milk or milk
with less lactose or cholesterol.
 Transgenic cows have been used to produce milk which
are richer in proteins and lower in fat.

35. (B) Quality
Herman, a transgenic bull carries a human gene for
Lactoferrin (gene responsible for higher iron content)
Pigs and cattle that have more meat on them.
Sheep that grow more wool.
Eggs can be made healthier with high quality protein
.
(C) Disease resistance
Disease-resistant livestock is not a reality just yet.
But there has been improvement in disease reduction in
animals.
Transgenic disease protection promises a long term cost
effective method of battling animal diseases.

36. 2. Medical Applications
A).Xenotransplantation
 Transplant organs may soon come from transgenic
animals.
• e.g. Transgenic pigs may provide the transplant organs
needed to alleviate the shortfall.
• Currently, xenotransplantation is hampered by a pig
protein that can cause donor rejection but research is
underway to remove the pig protein and replace it with
a human protein. For organ and tissue transplantation,
which is known as a "species of daughter cells “

37. B) Nutritional supplements and pharmaceuticals:
• Milk-producing transgenic animals are especially useful for
medicines.
• 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.
• This transgenic milk is a more nutritionally balanced
product than natural milk and could be given to babies or
the elderly with special nutritional or digestive needs.

38. D) Drug Development:
• Search for new drug targets.
• Validation of drug targets.
• Safety testing.
• Protein production.
C) 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.
• Finland produced a calf with a gene that makes the substance that
promotes the growth of red cells in humans.

39. 3. Industrial Applications
o Biosteel is an extraordinary new product that may be soon
used in bullet proof vests and in suture silk for stitching
wounds.
o Animals have been used as “Bioreactors” to produce
proteins. Genes for desired proteins are introduced via
transgenics to the target cells .
o The target cells are cloned and several such cells are raised
into adults.
o These adults may produce milk or eggs (due to the presence
of introduced gene rich in desired protein.

40. • 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.
• 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 microsutures,
and tennis racket strings.