Transgenic animals are produced through microinjection of DNA into fertilized eggs or embryonic stem cells. The document outlines various methods for producing transgenic animals, including DNA microinjection into pronuclei, retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. It also describes techniques for identifying transgenic animals, such as PCR and Southern blotting. Examples given of transgenic animals include Dolly the sheep and supermice.

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

10. MICROMANIPULATOR DEVICE
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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’
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12. Examples of transgenic animals:-
Sheep, Goats, Pigs, Cows, Rabbits, Rats,
Mice, Fish, Insects, etc…
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DOLLY
SUPERMICE/GAINT MICE
SILK MILK GOAT
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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.
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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.
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15. DNA-Microinjection Method
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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.
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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).
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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.
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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%).
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20. 2008-Apr-20 08:02 PM

21. TRANSGENE IS MICROINJECTED INTO
MALE PRONUCLEI.
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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.
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23. PRODUCTION OF TRANSGENIC MICE BY
RETROVIRAL VECTOR METHOD.
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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.
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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.
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26. STRUCTURE OF RETROVIRUES
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27. LIFE CYCLE OF RETROVIRUS
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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).
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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.
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30. Production of Transgenic Animals by
ES Cells:-
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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.
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34. Electropermeablization of Membrane
BEFORE
HYDROPHLIC HEADS
HYDROPHOBIC TAILS
AFTER HYDROPHLIC HEADS
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VOLTAGE APPLIED
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35. Cuvettes and Electroporation Machine
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36. Method of Electroporation
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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.
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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)
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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°
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[DNA Digestion buffer = EDTA + Nacl + Tris + Sodium dodecyl sulfate(SDS)]
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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)
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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)
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THERMAL CYCLER/THEROCYCLER/
PCR MACHINE/DNA AMPLIFIER
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42. P C R(POLYMERASE CHAIN REACTION)
42
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43. P C R CYCLE
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44. THERMAL STEPS IN PCR CYCLE
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45. DNA Sample introduced into
gel electrophoresis
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46. 4608-Apr-20 08:02 PM

47. SOUTHERN BLOTTING TECHNIQUE
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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
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49. Making of DOLLY(Female domestic sheep)
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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.
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SUPERMICE or
GAINT MICE
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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’
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SILK MILK GOAT
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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.
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SEA CORAL ZEBRA AQUERIUM FISH
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53. PRODUCTION OF TRANSGENIC FISH.
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54. 54
 HOUSING
 FEEDING
 VENTILATION
 LIGHTING
 SANITATION
 VETERNIARY CARE
 CLEANLINESS
MAINTANCE
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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.
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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
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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.
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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.
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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
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60. 60
THANK
YOU
08-Apr-20 08:02 PM