Transgenic goat and pig (A. Anbu Abubakkar Sidik).pptx

Transgenic goat and pig. These two topics are explained by the leading research article references

1. Transgenic Goat and Pig Submitted to, Dr. G. Ramanathan Assistant professor PG & Research Department of Microbiology SRI PARMAKALYANI COLLEGE OF ARTS AND SCIENCE ALWARKURICHI-627 412 SRI PARAMAKALYANI COLLEGE (PG & RESEARCH DEPARTMENT OF MICROBIOGY) ALWARKURICHI- 627412 Submitted by, A.Anbu Abubakkar Sidik II M.Sc. Microbiology PG & Research Department of Microbiology SRI PARMAKALYANI COLLEGE OF ARTS AND SCIENCE ALWARKURICHI-627 412 SEMESTER-IV BIOTECHNOLOGY

2. History Generating new and variable phenotypes via direct alteration of DNA sequences is an interesting idea that has sparked the curiosity of a wide spectrum of researchers over the past few decades. Based on significant efforts, tremendous advances have been achieved in animal genetics and reproductive physiology. These have enabled what is now known as the genome-editing revolution that can be applied to generate gene-edited animals including sheep and goats for various purposes About 40 years ago, a set of basic techniques were applied to sheep embryos with the desire to generate identical twins, multiplets, and chimeras.

3. In 1979 - The further development of these tools has led to the generation of identical individuals by embryo splitting - ( Willadsen ) In 1986 - Even the first cloned sheep prior to the famous Dolly from undifferentiated embryonic cells ( Willadsen ). in 1985 - The first report about the generation of transgenic farm animals (including sheep) via pronuclear injection (PNI) was published, announcing the first procedure for the production of transgenic farm animals About 10 years later, in 1996, success of cloning sheep from more differentiated embryonic cells has been reported ( Campbell et al., 1996 ). One year later, the same group announced unprecedented success by cloning the sheep Dolly from adult somatic cells ( Wilmut et al., 1997 ).

4. In 1985 – the first transgenic pigs were produced by injecting copies of human growth hormone genes into the pronuclei of newly fertilised eggs ( Hammer et al., 1985) First transgenic cloned goats in 1999 – (Baguisi et al., 1999 )

5. Introduction of foreign gene There are a number of methods to carry out the genetic modification of the animals a) injection of DNA construct directly into the pronuclei of zygotes Pronuclear injection is a technique used to create transgenic organisms by injecting genetic materials into the nucleus of a fertilized oocyte. This technique is commonly used to study the role of genes using mouse animal models. In order for pronuclear injection to be successful, the genetic material (typically linear DNA) Must be injected while the genetic material from the oocyte and sperm are separate (i.e., the pronuclear phase)

6. Pronuclear injection

7. Tool for genetic modification Include Sperm-mediated gene transfer viral vectors RNA interference Recombinases Transposons endonucleases

8. Retrovirus mediated transfer In this method the genes are removed from the genome of lentiviruses (a category of retroviruses) and replaced by the genes of interest. This is integrated into the chromosomes of the oocyte after its injection between the zone pellucida and the plasma membrane of oocyte that is arrested in the metaphase II of meiosis. It was carried out in pigs after application in cattle’s. Immediately following infection, the retrovirus produces a DNA copy of its RNA genome using its reverse transcriptase. the DNA copy of the viral genome, or provirus, integrates randomly the host cell genome, usually without deletions or rearrangements

9. Sperm mediated gene transfer A method highly efficient for the transgenic pig creation, whereby the in-vitro fertilization or insemination of the pigs was carried out with sperm previously mixed with DNA construct of interest. The genetic material is introduced into the sperm, which are used to fertilize eggs. The embryos are carried to term. The offspring may be transgenic.

10. Somatic cell nuclear transfer (SCNT) Technique in which the nucleus of a somatic (body) cell is transferred to the cytoplasm of an enucleated egg (an egg that has its own nucleus removed ). Once inside the egg, the somatic nucleus is reprogrammed by egg cytoplasmic factors to become a zygote (fertilized egg) nucleus. The egg is allowed to develop to the blastocyst stage, at which point a culture of embryonic stem cells (ESCs) can be created from the inner cell mass of the blastocyst

11. Embryonic stem cell technology (ES cell technology) As targeted integration is not achieved properly in other methods, it makes way for the development of Embryonic stem cells (ES cell technology) Embryonic stem cells come from a five to six day old embryo. They have the ability to form virtually any type of cell. Embryonic stem cells (ES cells) are harvested from the inner cells mass of blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes. However , this method has been successfully applied only in mice and for other species, a true Es cell that goes with germline is yet to be developed

12. Zinc finger nucleases Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes taking advantage of endogenous DNA repair machinery, these reagents can be used précised alter the genomes of higher organisms. A zinc finger is a small protein structural motif that is characterized by the coordination of the one or more zinc ions in order to stabilize the fold Zinc can be used to produce double-stand breaks (DSBs) in the DNA (see genetic recombinant) in the mutant allele, which will, in the absence of a homologous template, be repaired by non-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that the cut is clean and uncomplicated in some instances

13. Four classes of site specific endonucleases Meganucleases ZFNs TALENS CRISPRS

14. Analysis techniques PCR technique Analysis of transgene integration Analysis of mRNA production Analysis of protein expression Selectable marker is used to differentiate transformed from untransformed cells .

15. HOW GOATS ARE GENETICALLY MODIFIED?

16. Human hormone gene in livestock

17. xenotransplantation Transplantation of living cells, tissues, and organs from one species to another is known as xenotransplantation. Xenograft- is an organ transplanted from one species to another Human transplantation offers a potential treatment for end-stage organ failure, a significant health problem in parts of the industrialized world. Xenotransplants could save thousands of patients waiting for donated organs.

18. Pig as an animal organ donor Easy to breed Pathogen free pig breeds are available Pig organs are similar to that of size of humans Risk of infection is lower in non human primates Factors affecting xenotransplantation are : Longevity Size Environment Hormone and protein differences

19. Species Construct abbreviation Construct name Main trait References Goat mWAP / hLAtPA Mouse whey acidic protein/human longer-acting tissue plasminogen activator Therapeutic proteins in milk Ebert et al., 1991 b β Cas/ hFIX Bovine β-casein/human factor IX Therapeutic proteins in milk Huang et al., 1998 c β Cas/ hAT Caprine β- casein/human antithrombin Therapeutic proteins in milk Edmunds et al., 1998 c β Cas/ hG -CSF Caprine β-casein/human granulocyte colony-stimulating factor Therapeutic proteins in milk Ko et al., 2000 c β Cas/ hLF Caprine β- casein/human lactoferrin Therapeutic proteins in milk Zhang et al., 2008a b α S1Cas/ hLZ Bovine α s1-casein/human lysozyme Therapeutic proteins in milk Maga et al., 2003 c β Cas/ hBChE Caprine β- casein/human butyrylcholinesterase Therapeutic proteins in milk Huang et al., 2007 bBLG / rSCD Bovine β-lactoglobulin/rat stearoyl- coa desaturase Alteration of milk composition Reh et al., 2004 CMVp /cTLR2 Cytomegalovirus promoter/caprine toll-like receptor 2 Disease resistance Deng et al., 2012b

20. The Hyperacute rejection (HAR) of porcine xenografts is one of the major constraints. Humans posses natural anti-pig antibodies that are specific for alpha(1,3)- galactosyl epitopes on pig cells. Gal-alpha (1,3)- Gal is the proteins on the surface of pig cells but not human ones. Attempts have been made to reduce the amount of this sugar molecule by expressing antibodies against it, inhibiting the enzyme that makes it (an enzyme called alpha-1,3-galactosyltransferase that is only present in pigs) or using additional enzymes to modify it. Most recently, two research groups have succeeded in completely knocking out the alpha- 1,3- galactosyltransferase gene, producing pigs that cannot make this sugar at all

21. Bio steel Goat Bio steel goat haver been genetically modified to produce the protein from golden orb weaver spider silk in their milk. The gene that codes for protein that spiders use for their silk was transferred through laboratory techniques into the goats genome .

22. Goats for silk production??

23. What makes spider silk special? The strongest naturally occurring fiber known. It is tougher than Kevlar. Elasticity is better than nylon The high biocompatibility Resistance to extreme temperature variations Why do we need spider silk? To produce, Bulletproof vests Improved airbags In the medical field For jaw surgery To develop artificial ligaments, tendons, special sutures for eyes, human skin.

24. Applications of animal transgenesis Applications of animal transgenesis may be divided into three 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 humans) to be used for human therapy, and To improve animal products for human consumption.

25. CRISPR-cas9

26. journals Frontiers Genetics - Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era International journal of molecu - Generating Cloned Goats by Somatic Cell Nuclear .Transfer—Molecular Determinants and Application to Transgenics and Biomedicine

27. videos https://www.youtube.com/watch?v=U3Z4u0DKbx0&t=5s https://www.youtube.com/watch?v=4YKFw2KZA5o https://www.youtube.com/watch?v=sUtExVjK2Gc&t=199s https://www.youtube.com/watch?v=JArLDYv0Qw4 https://www.youtube.com/watch?v=1XNy9vqFcCg https://www.youtube.com/watch?v=YkVVkzA7QpA&t=18s