Transgenic animals

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. 2 nd Year M. pharm Dept-PHARMACOLOGY PDEA’S S.G.R.S COP,SASWAD 1

2. CONTENT PRODUCTION APPLICATION MAINTENANCE 2

3. DEFINITIONS 3

4. WHAT IS TRANSGENIC ANIMALS? 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 DNA Microinjection Retrovirus-mediated gene transfer Embryonic stem cell-mediated gene transfer Cre -lox recombination technique Testis cell transplantation method Artificial chromosome mediated gene transfer Lentiviral transfer method 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 10

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

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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

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18. CRE-LOX RECOMBINATION TECHNIQUE CRE CREATE LOX LOCUS OF X-OVER P 1 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 lox P sequence’ Bacteriophage P1 used to introduce inducible deletions. 18

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20. TESTIS CELL TRANSPLANTATION METHOD 20

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22. ARTIFICIAL CHROMOSOME MEDIATED GENE TRANSFER 22

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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. A ctivated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells 26

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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 H uman 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