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1 WEL COME 29-Jun-20 12:32 PM

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2 Presented by:- Lingaraj.V.Anawal M Pharm – 1 ST SEM Department of Pharmacology H S K College of Pharmacy B.G.K TRANSGENIC ANIMALS 29-Jun-20 12:32 PM

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OUTLINE:-  INTRODUCTION  DEFINITION  PRODUCTION aDNA microinjection bRetroviral vector method cEmbryonic stem cell mediated gene transfer dElectroporationEP  IDENTIFICATION OF TARGET GENE IN TRANSGENIC ANIMAL aPCR bSouthern blotting  TRANSGENIC ANIMALS  MAINTENANCE  APPLICATION  CONCLUSION  REFERENCE 3 29-Jun-20 12:32 PM

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 INTRODUCTION:- THE FIRST TRANSGENIC ANIMAL  IN 1966 TEH PING LINDept 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 21 st Jan 1996 vol.151 PP333-337  IN 1974 RUDOLF JAENISCHPROFESSOR AT THE STALK INSTITUTE AND BEATRICE MINTZAMERCIAN EMBRYOLOGIST AT THE FOX CHASE CANCER CENTRE IN PHILADELPHIA USED THESE METHODS TO MAKE THE FIRST “TRANSGENIC MOUSE” 4 29-Jun-20 12:32 PM

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PROFESSOR OF BIOLOGY/AMERCIAN EMBRYOLOGIST 5 RUDOLF JAENISCH BEATRICE MINTZ 29-Jun-20 12:32 PM

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 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. 6 29-Jun-20 12:32 PM

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 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 EIGHT8 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. 7 29-Jun-20 12:32 PM

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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:-MOUSEMus 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. 8 29-Jun-20 12:32 PM

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MOLECULAR GENETICIST /BIOLOGIST 9 OLIVER SMITHIES MARIO RAMBERG CAPECCHI MARTIN JOHN EVANS 29-Jun-20 12:32 PM

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MICROMANIPULATOR DEVICE 10 29-Jun-20 12:32 PM

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 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’ 11 29-Jun-20 12:32 PM

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Examples of transgenic animals:- Sheep Goats Pigs Cows Rabbits Rats Mice Fish Insects etc… 12 DOLLY SUPERMICE/GAINT MICE SILK MILK GOAT 29-Jun-20 12:32 PM

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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. 13 29-Jun-20 12:32 PM

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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. 14 29-Jun-20 12:32 PM

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DNA-Microinjection Method 15 29-Jun-20 12:32 PM

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PRODUCTION OF TRANSGENIC ANIMAL Young virgin female mice4-5 weeks age superovulation Injecting of Pregnant mare’s serumF.S.H 2days later48hr’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. 16 29-Jun-20 12:32 PM

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Eggs are washed kept at 37°C in culture media. Eggs are observed under dissecting microscope to distinguish two pronuclei.malefemale 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 miceFoster mother. 17 29-Jun-20 12:32 PM

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Pseudo pregnant female miceFoster 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 HybridizationTail blot / PCR. 18 29-Jun-20 12:32 PM

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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 motherfoster 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. 19 29-Jun-20 12:32 PM

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TRANSGENE IS MICROINJECTED INTO MALE PRONUCLEI. 21 29-Jun-20 12:32 PM

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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. 22 29-Jun-20 12:32 PM

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PRODUCTION OF TRANSGENIC MICE BY RETROVIRAL VECTOR METHOD. 23 29-Jun-20 12:32 PM

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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. 24 29-Jun-20 12:32 PM

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Then retrovirus is injected into pre-embryo 8-cell embryo Retrovirus containing pre-embryo are returned to recipient Pseudo pregnant and allow to develop. 25 29-Jun-20 12:32 PM

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STRUCTURE OF RETROVIRUES 26 29-Jun-20 12:32 PM

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LIFE CYCLE OF RETROVIRUS 27 29-Jun-20 12:32 PM

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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. 28 29-Jun-20 12:32 PM

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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. 29 29-Jun-20 12:32 PM

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Production of Transgenic Animals by ES Cells:- 30 29-Jun-20 12:32 PM

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1 ES Cell Collection 2 Preparing to Inject a Blastocyst 3 Embryonic Stem Cells Injection into Blastocyst 31 29-Jun-20 12:32 PM

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EMBRYONIC STEM CELLS ARE INJECTION INTO BLASTOCYST 32 29-Jun-20 12:32 PM

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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. 33 29-Jun-20 12:32 PM

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Electropermeablization of Membrane BEFORE HYDROPHLIC HEADS HYDROPHOBIC TAILS AFTER HYDROPHLIC HEADS 34 VOLTAGE APPLIED 29-Jun-20 12:32 PM

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Cuvettes and Electroporation Machine 35 29-Jun-20 12:32 PM

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Method of Electroporation 36 29-Jun-20 12:32 PM

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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. 37 29-Jun-20 12:32 PM

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Extraction of gene from Transgenic Animal. Remove Digest 0.5-2mm of mouse tail in a polypropylene centrifuge tube containing 300-500µL0.3-0.5mL of DNA digestion buffer with 0.4-0.5mg of Proteinase KPK/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 alcohol25:24:1 38 29-Jun-20 12:32 PM

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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. TETrisEDTA 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 sulfateSDS 29-Jun-20 12:32 PM

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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:- aTTPthymidine triphosphate bdCTPdeoxycyctidine triphosphate cdATPdeoxyadenosine triphosphate ddGTPdeoxyguanosine triphosphate  Heat stable DNA polymerase:- aTaqisolated from bacterium thermus acquaticus bPfuPyococcus furiosus cVentThermococcus litoralis 40 29-Jun-20 12:32 PM

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STEPS INVOLVED IN PCR/EQUIPMENT DOUBLE STRANDED GENEDNA DENATURATION STAGE2min 94-98°C ANNEALING STAGE1min 40-60°C ELONGATION STAGE2min 72°C 1 CYCLE cycle repeats 41 THERMAL CYCLER/THEROCYCLER/ PCR MACHINE/DNA AMPLIFIER 29-Jun-20 12:32 PM

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P C RPOLYMERASE CHAIN REACTION 42 29-Jun-20 12:33 PM

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P C R CYCLE 43 29-Jun-20 12:33 PM

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THERMAL STEPS IN PCR CYCLE 44 29-Jun-20 12:33 PM

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DNA Sample introduced into gel electrophoresis 45 29-Jun-20 12:33 PM

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SOUTHERN BLOTTING TECHNIQUE 47 29-Jun-20 12:33 PM

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Eg:-TRANSGENIC ANIMALS  DOLLYfemale domestic sheep  Dolly is a 1 st 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 29-Jun-20 12:33 PM

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Making of DOLLYFemale domestic sheep 49 29-Jun-20 12:33 PM

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 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 29-Jun-20 12:33 PM

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 PRODUCTION OF BIOSTEELHigh strength fiber-based material  Montreal based Nexia Biotechnology Company in Canada implanted a single spider GenusAraneus 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 29-Jun-20 12:33 PM

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 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 29-Jun-20 12:33 PM

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PRODUCTION OF TRANSGENIC FISH. 53 29-Jun-20 12:33 PM

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54 HOUSING FEEDING VENTILATION LIGHTING SANITATION VETERNIARY CARE CLEANLINESS MAINTANCE 29-Jun-20 12:33 PM

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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. 55 29-Jun-20 12:33 PM

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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 56 29-Jun-20 12:33 PM

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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. 57 29-Jun-20 12:33 PM

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 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. 58 29-Jun-20 12:33 PM

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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-2 nd Edition.  DNA Science A FIRST COARSE 2 nd edition by DAVID A. MICKLOSGREG A. FREYER WITH DAVID A. CROTTY.  i Genetics A Molecular Approach by Peter J. Russell 2 nd editionIE  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 59 29-Jun-20 12:33 PM

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60 THANK YOU 29-Jun-20 12:33 PM