Transgenic animals

1. 1
WEL
COME
08-Apr-20 08:02 PM

2. 2
Presented by:- Lingaraj.V.Anawal
M Pharm – 1ST SEM
Department of Pharmacology
H S K College of Pharmacy B.G.K
TRANSGENIC
ANIMALS
08-Apr-20 08:02 PM

3. OUTLINE:-
 INTRODUCTION
 DEFINITION
 PRODUCTION
a)DNA microinjection
b)Retroviral vector method
c)Embryonic stem cell mediated gene transfer
d)Electroporation(EP)
 IDENTIFICATION OF TARGET GENE IN TRANSGENIC
ANIMAL
a)PCR
b)Southern blotting
 TRANSGENIC ANIMALS
 MAINTENANCE
 APPLICATION
 CONCLUSION
 REFERENCE
308-Apr-20 08:02 PM

4.  INTRODUCTION:-
THE FIRST TRANSGENIC ANIMAL?
 IN 1966 TEH PING LIN(Dept of Anatomy) AT THE
UNIVERSITY OF CALIFORNIA SCHOOL OF
MEDICINE, SAN FRANCISCO, SHOWED THAT
MOUSE EGGS CAN SURVIVE HAVING A SMALL
QUANTITIES OF LIQUID INJECTED INTO THEM
WITH FINE GLASS NEEDLE. (ARTICLE:- Microinjection
of mouse eggs Published in Science by AAAS on 21st Jan 1996
vol.151 PP=333-337)
 IN 1974 RUDOLF JAENISCH(PROFESSOR) AT
THE STALK INSTITUTE AND BEATRICE
MINTZ(AMERCIAN EMBRYOLOGIST) AT THE
FOX CHASE CANCER CENTRE IN
PHILADELPHIA USED THESE METHODS TO
MAKE THE FIRST “TRANSGENIC MOUSE”
408-Apr-20 08:02 PM

5. PROFESSOR OF BIOLOGY/AMERCIAN EMBRYOLOGIST
5
RUDOLF JAENISCH
BEATRICE MINTZ08-Apr-20 08:02 PM

6.  THEY INJECTED PURIFIED DNA FROM A
SIMIAN VIRUS (SV40) INTO ITS MOUSE
BLASTOCYST.
 WHEN THE RESULTING MICE WERE RAISED
TO ADULTHOOD (SV40) DNA IS DETECTED
IN THEIR TISSUES, SUGGESTING THAT THE
INJECTED DNA HAD INTEGRATED INTO
THEIR GENOME.
 IN 1980 JON GORDON AND FRANK RUDLE
AT YALE UNIVERSITY INTRODUCED A
CLONED GENE INTO FERTILIZED MOUSE
EGGS VIA MICROINJECTION AND
SHOWED STABLE INTEGRATION OF THE
INJECTED GENE INTO SOMATIC CELLS.
608-Apr-20 08:02 PM

7.  BUT WITHIN A YEAR THIS TECHNIQUE PROVED
THAT TRANSGENE NOT ONLY INTEGRATE INTO
THE HOSTS GENOME, BUT ALSO ARE EXPRESSED
AND PASSED ON TO OFFSPRINGS THROUGH
GERM CELLS.
 “MICE BECOME THE WORLD’S FIRST
TRANSGENIC ANIMAL”
 BUT IT TOOK ANOTHER EIGHT(8) YEARS BEFORE
TRANSGENIC MICE WERE DEVELOPED THAT
PASSED THE TRANSGENE TO THEIR OFFSPRING.
 GENETICALLY MODIFIED MICE WERE CREATED IN
1984 THAT CARRIED CLONED ONCOGENES.
 THE FIRST ANIMAL TO SYNTHESIZE TRANSGENIC
PROTEIN IN THEIR MILK WERE MICE IN 1987.
708-Apr-20 08:02 PM

8. KNOCKOUT ANIMAL
 THESE ARE THE ANIMALS IN WHICH A
SPECIFIC GENE HAS BEEN INACTIVATED
OR KNOCKED OUT BY REPLACING
EXISTING GENE OR BY INTRODUCTION OF
A FOREIGN DNA SEQUENCE.
EXAMPLE:-MOUSE(Mus musculus)
 THE FIRST RECORDED KNOCKOUT MICE
WERE CREATED IN 1989 BY MARIO
RAMBERG CAPECCHI, MARTIN EVANS, AND
OLIVER SMITHIES.(BY TURNING OFF
SPECIFIC GENE)
 IN 2007 THEY WERE AWARDED NOBEL
PRIZE IN PHYSIOLOGY OR MEDICINE.
808-Apr-20 08:02 PM

9. MOLECULAR GENETICIST /BIOLOGIST
9
OLIVER SMITHIES
MARIO RAMBERG CAPECCHI
MARTIN JOHN EVANS08-Apr-20 08:02 PM

10. MICROMANIPULATOR DEVICE
1008-Apr-20 08:02 PM

11.  DEFINITION OF TRANSGENIC
ANIMAL:-
‘Transgenic animal is one whose genome has
been altered by the transfer of gene or genes
from another species or breed’
OR
‘Transgenic animal is one that carries a foreign
gene that has been deliberately inserted into its
genome’
OR
‘Transgenic animals is one containing
recombinant DNA molecules in its genome that
were introduced by intentional human
intervention’
1108-Apr-20 08:02 PM

12. Examples of transgenic animals:-
Sheep, Goats, Pigs, Cows, Rabbits, Rats,
Mice, Fish, Insects, etc…
12
DOLLY
SUPERMICE/GAINT MICE
SILK MILK GOAT
08-Apr-20 08:02 PM

13. PRODUCTION OF TRANGENIC
ANIMALS
 Transgenic animals can be produced by
following methods:-
 DNA Microinjection/Pronuclear
Microinjection.
 Retrovirus – Mediated Gene Transfer.
 Embryonic Stem Cell-Mediated Gene
Transfer.
 Electroporation/Electrotransfer.
1308-Apr-20 08:02 PM

14. DNA Microinjection or
Pronuclear Microinjection.
Definition:-
 Microinjection is a technique of delivering
foreign DNA into a living cell (a cell, egg,
embroy of animals) through a glass
micropipette.
 Microinjection (gene transfer) is most frequently
& commonly used technique for production of
transgenic animals.
1408-Apr-20 08:02 PM

15. DNA-Microinjection Method
1508-Apr-20 08:02 PM

16. PRODUCTION OF TRANSGENIC ANIMAL
Young virgin female mice(4-5 weeks age)
superovulation
Injecting of Pregnant mare’s serum(F.S.H)
2days later(48hr’s)
Injecting of Human Chorionic Gonadotropine
Hormone (H.C.G.H)
30-35 eggs produced (instead of 5-10 eggs)
Mated with male mice (female is sacrificed after 22hrs)
Oviducts are removed into buffer solution.
Oviducts are dissected to release fertilized eggs.
1608-Apr-20 08:02 PM

17. Eggs are washed & kept at 37°C in culture media.
Eggs are observed under dissecting microscope to
distinguish two pronuclei.(male&female)
1.2 PL of buffer containing cloned plasmid DNA is
injected into male pronuclei (microinjection needle)
Eggs with transgene kept overnight in incubator OR
culture media to develop.
Eggs are implanted micro-surgically into oviduct of
Pseudo pregnant female mice(Foster mother).
1708-Apr-20 08:02 PM

18. Pseudo pregnant female mice(Foster mother) deliver
pups after 3 weeks of implantation.
For identification of transgenic animals DNA from
small piece of tail can be assayed by southern blot
Hybridization(Tail blot) / PCR.
1808-Apr-20 08:02 PM

19. TRANSGENIC ANIMALS
 Less than 5% of
the Microinjected
Fertilized eggs
Become transgenic
progeny.
 In this method none of the steps will give 100% efficient for any animal to
develop into transgenic animal.
 In case of mouse of 100% injected only 60-70% of fertilized eggs will
Survive by microinjection procedure, same of about 60-70% of them
implanted into recipient mother(foster mother) to develop into pups, and
only <20% of them are live born & only <5% are transgenic progeny.
 Among 1000 fertilized eggs only 30-50 transgenic pups may be
produced (3%-5%).
1908-Apr-20 08:02 PM

20. 2008-Apr-20 08:02 PM

21. TRANSGENE IS MICROINJECTED INTO
MALE PRONUCLEI.
2108-Apr-20 08:02 PM

22. RETRO VIRUES MEDIATED GENE TRANSFER.
OR
RETROVIRAL VECTOR METHOD.
 A retrovirus is a virus that carries its genetic
material in the form of RNA rather than DNA.
 Retrovirus used as vectors to transfer genetic
material into the host cell, resulting in a chimera.
 This can be best be done at 4-16 cells stage
embryos.
2208-Apr-20 08:02 PM

23. PRODUCTION OF TRANSGENIC MICE BY
RETROVIRAL VECTOR METHOD.
2308-Apr-20 08:02 PM

24. Method of Production:-
Gene of interest is isolated using Restriction
Enzyme.
Vector is a strand of RNA from a retrovirus &
Vector is isolated & cut from the Restriction
Enzyme.
Target gene is inserted into the vector using DNA
Ligase.
The retrovirus contain nucleic acids from
different organism.
2408-Apr-20 08:02 PM

25. Then retrovirus is injected into pre-embryo
(8-cell embryo)
Retrovirus containing pre-embryo are returned to
recipient Pseudo pregnant and allow to develop.
2508-Apr-20 08:02 PM

26. STRUCTURE OF RETROVIRUES
2608-Apr-20 08:02 PM

27. LIFE CYCLE OF RETROVIRUS
2708-Apr-20 08:02 PM

28. Embryonic Stem Cell-Mediated Gene
Transfer
 Embryonic-steam cell mediated gene transfer is
one of the method which involve the introduction
of gene into the embryonic steam cells
(ES cells).
2808-Apr-20 08:02 PM

29. PRODUCTION
 Transgenic animals can be created by
manipulating embryonic stem cells.
 ES cells are obtained from the inner cell
mass of blastocyst.
 Transgene is incorporated in the ES cells by:-
 By Microinjection.
 By Retrovirus.
 By Electroporation/Electropermeabilization.
• Transgenic stem cells are grown in vitro.
• Then they are inserted into a blastocyst and
implanted into a hosts uterus to grow
normally.
2908-Apr-20 08:02 PM

30. Production of Transgenic Animals by
ES Cells:-
3008-Apr-20 08:02 PM

31. 1 ES Cell Collection 2 Preparing to Inject a Blastocyst
3 Embryonic Stem Cells Injection into Blastocyst 3108-Apr-20 08:02 PM

32. EMBRYONIC STEM CELLS ARE
INJECTION INTO BLASTOCYST
3208-Apr-20 08:02 PM

33. Electroporation /Electropermeablization
 Electroporation/Electropermeabilization is a
microbiology technique in which an electrical field
is applied to cells in order to increase the
permeability of the cell membrane & allowing DNA,
Chemicals, Drugs to enter inside the cells.
3308-Apr-20 08:02 PM

34. Electropermeablization of Membrane
BEFORE
HYDROPHLIC HEADS
HYDROPHOBIC TAILS
AFTER HYDROPHLIC HEADS
34
VOLTAGE APPLIED
08-Apr-20 08:02 PM

35. Cuvettes and Electroporation Machine
3508-Apr-20 08:02 PM

36. Method of Electroporation
3608-Apr-20 08:02 PM

37. IDENTIFICATION OF TARGET GENE IN
TRANSGENIC ANIMALS.
 Transgenic animals are identified by:-
 PCR (POLYMERASE CHAIN REACTION)
Technique.
 Southern Blotting.
 PCR:- PCR is laboratory technique which is widely used
to make multiple copies of a segment of DNA.
 It is very precise and can be used to amplify or copy a
specific DNA target from a mixture of DNA molecule.
 SOUTHERN BLOTTING:- It is one of the method or
procedure which is used to identify/detect a specific
DNA sequence in DNA samples.
3708-Apr-20 08:02 PM

38. Extraction of gene from Transgenic
Animal.
Remove & Digest 0.5-2mm of mouse tail in a polypropylene
centrifuge tube containing 300-500µL(0.3-0.5mL) of DNA
digestion buffer with 0.4-0.5mg of Proteinase K(PK)/ml of
digestion buffer in a 1.5ml in a polypropylene tube.
Incubate at 50-55°C overnight with gently shaking
(mechanical agitation)
Add 0.7-1ml of 100% of ethanol
OR
Neutralized phenol/chloroform/iso-amyl alcohol(25:24:1)
3808-Apr-20 08:02 PM

39. Centrifuge at top speed for 5 min or 16000xg for 30 min.
pour/remove out ethanol.
Wash out DNA pellets by adding 1ml 70% of ethanol
into the tubes & then Re-centrifuge at a top speed for
5min.
Pour out ethanol from the tubes, immediately dissolve
those DNA pellets with 100-300µl TE-buffer solution
without air drying. (TE=Tris&EDTA)
Place the tubes for at 55-60°C for approximately 2hrs
with lids open to let the extra ethanol to evaporate.
The store the DNA sample at 4°C to -20°
39
[DNA Digestion buffer = EDTA + Nacl + Tris + Sodium dodecyl sulfate(SDS)]
08-Apr-20 08:02 PM

40. PCR TECHNIQUE AND REQUIREMENTS
 The PCR is carried out in in vitro.
 It utilizes :- DNA preparation containing the desired
segment of DNA to be amplified.
 Two nucleotide primers of specific to 3’ borders.
 Four deoxynucleoside triphosphates:-
(a)TTP(thymidine triphosphate)
(b)dCTP(deoxycyctidine triphosphate)
(c)dATP(deoxyadenosine triphosphate)
(d)dGTP(deoxyguanosine triphosphate)
 Heat stable DNA polymerase:-
(a)Taq(isolated from bacterium thermus
acquaticus)
(b)Pfu(Pyococcus furiosus)
(c)Vent(Thermococcus litoralis)
4008-Apr-20 08:02 PM

41. STEPS INVOLVED IN PCR/EQUIPMENT
DOUBLE STRANDED GENE(DNA)
DENATURATION STAGE(2min)
(94-98°C)
ANNEALING STAGE(1min)
(40-60°C)
ELONGATION STAGE(2min)
(72°C) (1 CYCLE)
(cycle repeats)
41
THERMAL CYCLER/THEROCYCLER/
PCR MACHINE/DNA AMPLIFIER
08-Apr-20 08:02 PM

42. P C R(POLYMERASE CHAIN REACTION)
42
08-Apr-20 08:02 PM

43. P C R CYCLE
4308-Apr-20 08:02 PM

44. THERMAL STEPS IN PCR CYCLE
4408-Apr-20 08:02 PM

45. DNA Sample introduced into
gel electrophoresis
4508-Apr-20 08:02 PM

46. 4608-Apr-20 08:02 PM

47. SOUTHERN BLOTTING TECHNIQUE
4708-Apr-20 08:02 PM

48. Eg:-TRANSGENIC ANIMALS
 DOLLY(female domestic sheep)
 Dolly is a 1st mammal cloned from an adult somatic cell
using the process of nuclear transfer.
 Experiment is carried out by Sir Ian Wilmut, Keith Campbell
& colleagues at the Roslin institute in collaboration with
biotechnology company PPL therapeutics in Edinburgh
Scotland.
48
DOLLY
08-Apr-20 08:02 PM

49. Making of DOLLY(Female domestic sheep)
4908-Apr-20 08:02 PM

50.  SUPERMICE OR GAINT MICE
 In 1982 Ralph Lawrence Brinster & Colleagues at
the university of Pennsylvania school of veterinary
medicine successfully injected the gene encoding
rat GH into mouse embryo.
50
SUPERMICE or
GAINT MICE
08-Apr-20 08:02 PM

51.  PRODUCTION OF BIOSTEEL(High strength
fiber-based material)
 Montreal based Nexia Biotechnology Company in Canada
implanted a single spider (Genus=Araneus) gene into the egg
cells of lactating Nigerian Dwarf Goat. Their cloning led to the
birth of the first ‘silk milk goat’
51
SILK MILK GOAT
08-Apr-20 08:02 PM

52.  ORNAMENTAL FISH PRODUCTION
 The Glofish was common zebra (Danio rerio)
aquarium fish which was fluorescent red in color due
to the insertion of red fluorescent sea coral gene.
52
SEA CORAL ZEBRA AQUERIUM FISH
08-Apr-20 08:02 PM

53. PRODUCTION OF TRANSGENIC FISH.
5308-Apr-20 08:02 PM

54. 54
 HOUSING
 FEEDING
 VENTILATION
 LIGHTING
 SANITATION
 VETERNIARY CARE
 CLEANLINESS
MAINTANCE
08-Apr-20 08:02 PM

55. APPLICATION
 Industrial importance:-
 Toxicity sensitive transgenic animals to test
chemicals.
 Spider silk in milk of goat.
 For Pharmaceutical testing and development.
 As bioreactors to produce pharmacologically
important proteins.
 Medical importance:-
 Disease model.
 Xenotransplantation. /Xenografting
 Bioreactor for pharmaceutical.
5508-Apr-20 08:02 PM

56.  Agricultural importance:-
 Disease resistance animals.
 For improving quality and quantity of milk, meat,
eggs, and wool production etc…
 Biological function of specific genes.
 To develop animal model for disease
of humans or animals.
 To produce therapeutic products,
and vaccines.
 Biological screening etc…
 Vaccine safety e.g.:- polio vaccine
5608-Apr-20 08:02 PM

57. CONCLUSION
 Transgenic Technology is a field that is under
constant evolution.
 Transgenic animals are now-a-days used for
screening of many drugs.
 Transgenic animals reduce number of
experimental animals during testing.
 Many of transgenic animals have been
successfully created for a variety of purposes
and prospects are enormous.
 It also hold a great potential in many field
including agriculture, medicine, and industry.
5708-Apr-20 08:02 PM

58.  With a proper research and careful use the
transgenic animals can go a long way in solving
several problems for which science doesn’t have
a solution till now.
5808-Apr-20 08:02 PM

59. REFERENCE:-
 Screening Methods in Pharmacology N.S.Parmar
Shiva Prakash Narsosa Publishing House.
 Molecular Biotechnology (Principles and Applications of
Recombinant DNA) by Bernard R. Glick and Jack J.
Pasternak-2nd Edition.
 DNA Science A FIRST COARSE 2nd edition by DAVID A.
MICKLOS,GREG A. FREYER WITH DAVID A. CROTTY.
 i Genetics A Molecular Approach by Peter J. Russell 2nd
edition(IE)
 https://www.future-science.com/doi/full/10.2144/000112255
 https://www.jax.org/jax-mice-and-services/customer-
support/technical-support/genotyping-resources/dna-
isolation-protocols
5908-Apr-20 08:02 PM

60. 60
THANK
YOU
08-Apr-20 08:02 PM