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1. Transgenic animals: production maintenance and applications Presented By- Mohd Monish M.Pharm 1 st Semester Student Department of Pharmacology, SPER Jamia Hamdard

2. Presentation Outcome Transgenic Animals…………………………………………………………………………….03 Transgenesis……………………………………………………………………………………..04-06 Methods for producing Transgenic animals………………………………………..07-17 Transgenic Animal Detection………………………………………………………………18-19 Maintenance of Transgenic animals…………………………………………………….20 Applications………………………………………..………………………………………………21-23 Examples of Transgenic animals…………………………………………………………..24-25 Ethical Issue……………………………………….……………………………………………….26 References………………………………………..………………………………………………….27 2

3. Transgenic Animals A transgenic animal is one whose genome has been altered by transfer of gene or genes from another species or breed. The photo shows two transgenic mice positioned either side of a plain mouse. The transgenic mice have been genetically modified so that they carry a green fluorescent protein which glows green under blue light.  3

4. Transgenesis Process of introducing foreign or exogenous DNA into an animals genome is called transgenesis. Transgenesis is the process of introducing an exogenous gene called a transgene into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. 4

5. Why we use Transgenic Technology ? Transgenic animals have been instrumental in providing new insights and mechanisms of: Development and developmental gene regulation Action of oncogenes Complex cell interactions within the immune system The transgenic technology offers exciting possibilities for generating precise animal models for human genetic diseases and for producing large quantities of economically important proteins by means of genetically engineered farms animals. 5

6. Why Mice were chosen for Transgenic studies? Easily housed, maintained and adapt well to new surroundings. Reproduce quickly, have short lifespan of 2-3 years Relatively inexpensive Mild tempered and docile Medical Trials are uniform Mimic human body 6

7. Methods for Producing Transgenic Animals: DNA Microinjection Embryonic Stem Cell-Mediated Gene Transfer Retrovirus mediated gene transfer Transfection of Gametes Nuclear Transfer Method Artificial Chromosome Mediated Gene Transfer Testis cell transplantation method 7

8. DNA Microinjection DNA microinjection is the dominating technique leading to random integration of a transgene  via  the introduction of DNA into the pronucleus of a developing zygote. 8

9. Cont.. Fertilized eggs are collected from pregnant female mouse Fertilized eggs are held under suction at the end of a pipette The transgene is microinjected into one of the pronuclei with fine needle The eggs are then implanted into oviduct of pseudopregnant female The transgene will be incorporated into chromosomal DNA of about 10%to 30% of the spring 9

10. Embryonic Stem Cell-Mediated Gene Transfer In 1981, the term embryonic stem cells (ES cells) were used to denote a cell line isolated directly from mouse embryos while, the term embryonal carcinoma cells (EC) were derived from teratocarcinomas. Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes. 10

11. Introduction of foreign DNA into embryonic stem cell by electroporation or microinjection Desired genes in cells can be identified by selection using marker gene or PCR Transfected cells can be cultivated, Introduced into blastocyst by microinjection Implanted into foster mother Transgenic founder mice are produced 11 Steps Involved in Embryonic stem cell mediated gene transfer technique

12. 12 Fig: Embryonic Stem Cell-Mediated Gene Transfer

13. Retroviral gene delivery method Transgenic mice produced by retroviral transduction of male germ line stem cells. A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. Retroviruses used as vectors to transfer genetic material into the host cell, resulting into a generation of chimera (an organism consisting of tissues or parts of diverse genetic constitution). Chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born. 13

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15. Nuclear Transfer Method In this method, the transgenic s heep were produced by nuclear transfer of fetal somatic cells. Donor karyoplasts 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. 15

16. 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 represents latest generation of vectors which have the great advantage of large insert size. This method succeeded in mice and rabbits 16

17. Testis cell transplantation method Testis cell transplantation method is as follows:  A single-cell suspension is produced from a fertile donor testis.  The cells can be cultured  Microinjected into the lumen of seminiferous tubules of an infertile recipient mouse. Only a spermatogonia stem cell can generate a colony of spermatogenesis in the recipient testis. When 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. Mating the recipient male to a wild-type female  Produces progeny, which carry donor genes 17

18. Transgenic Animals Detection Dot blot technique: Dot blot is  a technique for detecting, analyzing, and identifying proteins, similar to the western blot technique, but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate. PCR Technique: The technique for detecting both foreign and host specific DNA sequences inside nuclei and chromosomes of single cells of transgenic animals with the help of polymerase chain reaction (PCR). 18

19. Analysis of transgene integration using restriction endonucleases : The integration of transgene into the genome is confirmed by southern hybridisation of genomic DNA extracted from transgenic individuals, DNA digested with suitable restriction enzyme prior to electrophoresis. Analysis of mRNA production: A high purity RNA preparation is obtained from transgenic individuals and subjected to northern hybridisation, which provides additional information on transcript size as well. 19

20. Maintenance of Transgenic animals Housing Feeding Ventilation Lighting Sanitation Routine management practices 20

21. Application of animal transgenesis may be divided into 3 major categories To obtain information on gene function and regulation as well as on human diseases. To obtain high value products (recombinant pharmaceutical proteins and xeno - organs for human) to be used for human therapy. To improve animal products for human consumption. All these applications directly or not are in someway related to human health. 21

22. Applications of Transgenic animals As disease model : Transgenic animals are produced as disease models (animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied) such as Alzheimer's, cancer, AIDS. Blood replacement: Transgenic swine are used to produce human hemoglobin. The protein obtained from transgenesis could be purified by using porcine blood which is similar to human hemoglobin. Xenotransplantation : Xenotransplantation, or heterologous transplant, is the transplantation of living cells, tissues or organs from one species to another. Such cells, tissues or organs are called xenografts or xenotransplants.   e.g. Pig organs (particularly kidney and heart) function very similarly to human kidneys and hearts. 22

23. Disease resistance : To produce disease resistant animals, such as influenza resistant pigs. Industrial application : Toxicity sensitive transgenic animals to test chemicals. Vaccine safety : Transgenic mice are being used in testing the safety of Vaccines before they are used in humans e.g. Polio vaccine Chemical safety testing : Transgenic animals are made to carry the genes which make them more sensitive to the toxic substance then the non transgenic ones. They are then exposed to toxic substance and effect are studied. 23

24. Some examples of Transgenic animals Transgenic Cow: The name of the first transgenic cow is Rosie and it was developed in 1997. They are mainly developed with two objectives- increased milk production and molecular farming. Transgenic Rabbit: The first transgenic rabbit (Alba) was successfully produced in 1985. Used as a model for cardiovascular disease, AIDS, Cancer. 24

25. Transgenic Fish Genetic transfer has been done in various fishes such as common carp, Atlantic salmon, etc. Transgenic salmon was the first genetically modified animal for food production. Salmon can grow 6 times faster than wild type as they have extra copies of growth hormone gene. Transgenic monkey ANDI, the first transgenic monkey born on October 2nd ,2000, has GFP gene. Glow in dark monkey also have GFP( green fluorescent protein) and glow under UV light. Model for studying autism, Alzheimer’s etc. 25

26. Ethical Issues Religious concerns : For Hindu, Muslim or Sikh it would be forbidden to eat food containing genetic material from the animals whose flesh is forbidden. Animal welfare : In vitro procedures might lead to increase gestation length , body weight and perinatal loss and anomalies. Microinjection increase embryonic and fatal losses. Economical aspects : Actions carried out by the researchers do not have any more like simple goal research of the truth but well a race with the property of living and its patentability which may produce colossal benefits. Environmental effects : Release of transgenic animals into environment may outcompete the natural population which effect the natural biodiversity and ecosystem. Other concerns : Violating animals bodily integrity, violating animals dignity/ Intrinsic value. 26

27. References Bernard et al. Molecular Biotechnology, Principles and Applications of Recombinant DNA, 4 th ed. ASM Press Washington DC, 2010;21: 845-861 Heiner, Christine, Animal Biotechnology 1, Embryo Transfer Technologies, Springer,2018;07: 167-178. Manmohan, Neeraj, Transgenic Animals: Production and Application, IJPSR (2010), Vol.1 (9): 12-22. Ingrid Moen et al.,  BMC Cancer,  12/21 (2012), 1-10. 27

28. Thank You 28