DEFINITIONS, REASONS FOR TRANSGENIC ANIMALS, PRODUCTION, DNA Microinjection method , RETROVIRUS MEDIATED GENE TRANSFER, EMBRYONIC STEM CELL MEDIATED GENE TRANSFER, CRE-LOX RECOMBINATION TECHNIQUE, TESTIS CELL TRANSPLANTATION METHOD, ARTIFICIAL CHROMOSOME MEDIATED GENE TRANSFER , NUCLEAR TRANSFER METHOD, APPLICATONS

1. TRANSGENIC ANIMALS
GUIDED BY:-
Mrs. JAGTAP P.N.
HOD OF PHARMACOLOGY
PDEA’S S.G.R.S. COP, SASWAD
PREPARED BY:-
Mr. MEMANE PRANESH P.
2nd Year M. pharm
Dept-PHARMACOLOGY
PDEA’S S.G.R.S COP,SASWAD 1

2. CONTENT
 PRODUCTION
APPLICATION
 MAINTENANCE
2

3. Transgenic animals are the animals that have a foreign gene
deliberately inserted into their genome by genetic engineering.
Transgenic animals have genetic material from another species.
The transgenic animal’s germ–cell DNA can be transmitted from
one generation to another.
DEFINITIONS
3

4. • It is technique used to incorporate exogenous gene into the animal genome by
genetic engineering technique so that these gene can be inherited and
expressed by off-springs.
• For studying gene function.
• Create models for human disease study .
• It is fastest growing technique .
4

5. • Animals that gains new genetic information or whose gene is manipulated-----
Transgenic animals
• Gene that is transferred ---TRANSGENE
5

6. WHICH ANIMALS CAN BE USED AS
MODELS?
RATS
MICE
RABBITS
FISH
SHEEP
COWS
PIGS
AND MANY MORE……
6

7. REASONS FOR TRANSGENIC ANIMALS
 AS DISEASE MODELS --- Genetically it is manipulated to exhibit
disease symptoms so that effective treatment can be studied.
 FOR ECONOMIC PURPOSE --- For economic purpose it is carried out
to get good quality of product and yield.
7

8. PRODUCTION
1) DNA Microinjection
2) Retrovirus-mediated gene transfer
3) Embryonic stem cell-mediated gene transfer
4) Cre-lox recombination technique
5) Testis cell transplantation method
6) Artificial chromosome mediated gene transfer
7) Lentiviral transfer method
8) Nuclear transfer method
8

9. DNA Microinjection method
• Firstly this method was used to prepare transgenic animal.
• Super mouse was developed using it.
• Human growth hormone was incorporated in the mouse and a transgenic
animal was developed using this technique.
9

10. STEPS
A female animal is super ovulated and its egg
are collected.
Eggs are fertilised IN-VITRO
Transgene containing solution is injected into
the male pro-nucleus using a micropipette
Eggs with transgenes are kept overnight in an
incubator to develop to a 2 stage cell
The eggs are then implanted into the uterus of
a pseudo-pregnant female i.e. foster mother.
10

11. 11

12. RETROVIRUS MEDIATED GENE
TRANSFER
• It is used as a vector.
It carries a genetic material.
It is in RNA form.
RETROVIRUS
12

13. STEPS
RNA virus vector containing a genetic
material infects the host cell of animal
model.
RNA is reversed transcripted into
double-stranded DNA into the
cytoplasm of the host cell.
This technique forms
CHIMERA i.e. a single
organism with different
genotypes in it. 13

14. 14

15. EMBRYONIC STEM CELL MEDIATED
GENE TRANSFER
 EMBRYONIC STEM CELLS should be totipotent in nature.
{totipotent i.e. they should divide into any type of specialised cell.}
 In this technique the transgenic off-springs do not require to be live ,the
transgene is seen in the cell stage only.
15

16. STEPS
Embryonic stem cells ae collected from the
inner cell mass of a blastocyst.
Transgene incorporated into the blastocyst
through—
1.Microinjection method
2.By a retro virus
3.By electroporation
Transgenic stem cells are grown IN-VITRO
16

17. 17

18. CRE-LOX RECOMBINATION TECHNIQUE
• CRE CREATE
• LOX LOCUS OF X-OVER P1 BACTERIOPHAGE
• Cre-lox technique is a site-specific recombinase technology, used to carry out
addition, deletions, insertions, translocations and inversions at specific sites.
• This site is termed as loxP sequence’
• Bacteriophage P1 used to introduce inducible deletions.
18

19. 2 components
CRE-RECOMBINASE
It recognise and splice
specific DNA sequences.
LoxP sites
13bp+8bp+13bp
2 flanking palindromic
sequences at end and
spacer in between.
19

20. TESTIS CELL TRANSPLANTATION METHOD
A) A single-cell suspension is produced from a fertile donor testis.
B) The cells can be cultured
C) Microinjected into the lumen of seminiferous tubules of an infertile recipient mouse.
D) When recipient testis cells carry a reporter transgene that allows the cells to be stained
blue, colonies of donor cell-derived spermatogenesis are identified easily in recipient
testes as blue stretches of tubule.
E) Mating the recipient male to a wild-type female.
F) Produces progeny, which carry donor genes. 20

21. 21

22. ARTIFICIAL CHROMOSOME MEDIATED GENE
TRANSFER
A group of nuclei injected with transgene DNA, the eggs are
transferred in medium of incubation and visual evaluation within next
few hours.
An individual animal develops after receiving the transgene DNA is
referred as founder of a new transgenic lineage.
Yeast Artificial Chromosomes (YACs) transgenic mice are generated
by using pronuclear microinjection and large insert size is possible.
22

23. 23

24. It overcome
previous
limitations of
viral mediated
gene transfer
It contains the
silencing of the
transgenic locus
and low
expression levels
It includes generation of
transgenic cattle by
lentiviruses requires
microinjection into the
oocytes
This study
employed
lentiviral based
vectors.
24

25. ADVANTAGES
These vectors have several advantages compared to
the conventional retro-vectors
• They can infect non-diving cell
• Can carry large amounts of transgene~10kb
• can show stable expression in the tissue
• The technique was successful in showing about
100 fold increases in the level of transgenesis .
25

26. NUCLEAR TRANSFER METHOD
Nuclear transfer of foetal somatic cell.
Donor karyoplast were obtained from a primary foetal somatic cell
line derived from a 40-day transgenic female foetus produced by
artificial insemination of a non-transgenic adult female with semen
from a transgenic male. Live offspring were produced with two
nuclear transfer procedures.
Oocytes at the arrested
metaphase II stage were
enucleated, electrofused
with donor somatic cells,
and simultaneously
activated.
Activated in vivo oocytes
were enucleated at the
telophase II stage,
electrofused with donor
somatic cells 26

27. 27

28. ADVANTAGE
The nuclear transfer application may be more
useful and beneficial for agricultural is the
ability to efficiently produce a large number of
identical offspring derived from a particular
mating.
28

29. FUNCTIONAL IMPERAVTIVES
• Proper sanitation is required.
• Materials should be durable, impervious, resistant to the water and chemicals.
• All sterilised equipment should be used.
• Good quality air at the appropriate temperature and humidity must be their.
• Security systems that limit access to authorised individuals only must be in place.
• Designated area should be provided to carry out all the animal procedures.
• Adequate storage space should be their for all cages and equipments that are not in
use.
MAINTENANCE
29

30. LOCATION
• It must be accessible to the reliable services, including water, electricity and the sewage
disposal.
• They should be such located that exhausted air does not enter the facility or the other
buildings.
• Incoming air and exhausting air should be treated properly.
• Animal feed and bedding must be stored in vermin-proof room.
• It should not be stored directly on the floor.
HOUSING
• Proper cages should be kept for the transgenic animals , they should be kept isolated from
others.
• Wooden cages are not allowed as they catches moisture.
• Male and female animals should not be kept together.
• Animal feed and bedding must be stored in vermin-proof room.
• It should not be stored directly on the floor.
30

31. ENVIRONMENT
• Equipment and activities that generate large amounts of noise should be kept isolated
from the rest of the animals.
• The frequency of alarm and bells in the animal facility should be selected in the
range that does not affect the animals. Mostly VISUAL ALARMS should be
preferred.
• Humidity should be maintained from 40-60% (+_5%)depending upon species.
• Air supplies should be 100% fresh and it should not recirculate within the facility.
• Exhaust duct should be fitted with the filters at the room level to reduce
accumulation of particulate matter. All the exhaust ducts should be tightly sealed.
31

32. • Especially in rodent animals lighting should be designed to provide at least 2
levels of intensity during light cycle.
• DIURNAL LIGHT cycles in animal holding room s should be controlled and
monitored centrally.
• The wavelength of light should simulate the wavelength of the sunlight as
closely as possible.
• The heating, ventilation and air conditioning (HVAC) should provide a
healthy and comfortable environment for animals.
• The temperature of each room should be controlled separately.
32

33. APPLICATONS
Human health: For the production of recombinant and biologically active
proteins in the mammary gland and this in turn could be used for the benefit of
mankind. This is called as “Gene Pharming”.
Proteins like anti-thrombin III (AT III), tissue plasminogen activator (TPA)
and á-antitrypsin have been derived from the mammary gland of transgenic
sheep and goats.
33

34. • Milk production: The animals could be made to secrete nutraceuticals in
milk that may have an impact over the growth of offspring.
They also secrete antibodies in their milk that give resistance against
several diseases like mastitis or to secrete antimicrobial peptides like lysozyme.
• Disease resistance: The most important application of transgenic technology
is the manipulation of MHC (Major Histocompatibility Complex) genes,
which influence the immune response and increase the disease resistance
capacity of livestock
34

35. •Tissue repair: Using induced pluripotent stem (iPS) cells were directly injected into the
vitreous of the damaged retina of mice, the stem cells engrafted into the retina, grow and
repaired the vascular vessels.
•Blood substitutes: Transgenic swine produce functional haemoglobin which has the
same oxygen binding capacity as that of normal human haemoglobin.
• Growth factor: Growth hormone and insulin like growth factors genes have been
expressed at different levels in transgenic cattle and salmon fish.
35

36. THANK YOU
36