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Transgenic animals.How we Produce transgenic animals
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- 4. Animals in Research Animationof Drug Development Process
- 5. z Animal Models •Mice • Rats • Zebrafish (3 month generation time, 200 progeny, complete embryogenesis in 120 hrs) • Dogs (lungs and cardiovascular system) • Cats • Pigs (PPL Therapeutics- delete a gene which causes hyperacute rejection of pig-to-human organ transplantation) • Primates (HIV and AIDs research, geriatric research) Animals in Research
- 6. z Alternatives toAnimal Models • Cell culture devices • Researchers use cell cultures and computer-generated models whenever possible, but this doesn’t work for looking at an organ or entire animal Animals in Research
- 7. z Regulation ofAnimal Research • The “Three Rs” • Reduce the number of higher species (cats, dogs, primates) used • Replace animals with alternative models whenever possible • Refine tests and experiments to ensure the most humane conditions possible Animals in Research
- 8. z Veterinary Medicineas Clinical Trials • Treatments for humans may also be useful for treatments with animals (e.g. the BRCA1 gene found in 65% of human breast tumors is similar to the BRCA1 gene in dogs) • Hyperthermia + radiation = more effective at killing tumors • Stimulation of cytokines for curing skin cancers Animals in Research
- 9. Bioengineering Mosquitoes toPrevent Malaria • Cloned in a gene that prevents the parasite from crossing the midgut; blocking the continuation of its life cycle • Developed an antibody that prevents the parasite from entering the mosquito’s salivary gland Animals in Research
- 10. Clones z Cloning • CreatingDolly • Limits to Cloning: The donor cell must come from a living organism • An organism is also shaped by its environment • The success rate for cloning is very low • Clones may be old before their time • The future of cloning: preservation of endangered animals, studying the effect of drugs etc on duplicates, improve agricultural production
- 11. Transgenic Animals z Pronuclearmicroinjection z Retrovirus-mediated transgenesis z Embronyic stem cell method z Sperm-mediated transfer
- 13. A pronucleus (pl. : pronuclei) denotes the nucleus found in either a sperm or egg cell duri the process of fertilization.
- 14. Retrovirus-mediated transgenesis z Intransgenic animal production, gene transfer efficiency is the limiting factor in transgenesis success rates. z Among many gene transfer systems developed to date, the retrovirus vector- mediated gene transfer system has been an matchless choice in gene transfer efficiency. z The most important features of retroviruses in regard to their use as vectors are the technical ease and effectiveness of gene transfer, due to their affinity and infectivity for certain target cells, leading to successful transgene incorporation. z Once cells are infected by retroviruses, the resultant viral DNA, after reverse transcription and integration, becomes a part of the host cell genome and is maintained for the life of the host cell. z In addition, it is believed that DNase hypersensitive regions are the preferred targets for retrovirus integration implying efficient expression of exogenous proviral genes even though the proviral copy number for each integration site is limited to a single copy. z Unlike DNA microinjection, integration of a viral gene does not seem to induce rearrangements of the host genome
- 15. z Improving AgriculturalProducts with Transgenics • Faster growth rates or shorter growth patterns (improve the product), more product • Increase nutritional content- lactoferrin • Turning the animals into efficient grazers • Transfer antimicrobial genes to farm animals Transgenic Animals
- 16. The majority ofsomatic ce are leukocytes (white blood which become present in in numbers in milk usually as response to a mastitis-cau pathogen - and a small num epithelial cells, which are m producing cells shed from udder when an infection oc
- 17. z Transgenic Animalsas Bioreactors • Biosteel otherwise known as spider silk, cloned into goat milk (“silkmilk” goats) • Goats reproduce faster than cows and are cheaper than cows • Hens also make good bioreactors in that they are cheap and a lot of eggs are produced at one time Transgenic Animals
- 18. Transgenic Animals as Bioreactors Researchersfrom the University of Wyoming have developed a way to incorporate spiders' silk-spinning genes into goats, allowing the researchers to harvest the silk protein from the goats’ milk for a variety of applications. For instance, due to its strength and elasticity, spider silk fiber could have several medical uses, such as for making artificial ligaments and tendons, for eye sutures, and for jaw repair. The silk could also have applications in bulletproof vests and improved car airbags. z Why is the military interested in goat milk spider silk? z The protein's antimicrobial capabilities also allows its for wound patch production. Safety equipment used in the military, sports, and automotive industries can benefit from this by incorporating them on protective gears, helmets, body and vehicle armors, cables, bulletproof vests, tires, and car airbags
- 19. z Knock-outs: ASpecial Case of Transgenesis • A specific gene is disrupted or removed such that it is not expressed • Procedure: DNA is modified, it is added to embryonic stem (ES) cells, where it undergoes homologous recombination. • The modified ES cells are then introduced into normal embryo. The embryo is implanted in an incubator mother. • The offspring is a chimera. It may take several generations of crossbreeding are required to produce animals that are complete knock-outs. • Breast cancer mouse Transgenic Animals
- 21. Antisense or RNAInterference Approaches to Decrease Gene Expression z Silencing of genes z 1. In an antisense gene construct delivered into the plant genome, the gene to be downregulated (that is, silenced) is essentially put in “backwards” into a plant transformation vector. z When the backwards gene is transcribed, the messenger RNA (mRNA) produced from the transgene interferes with the translation of complementary mRNA of the gene to be silenced in the plant or pest into protein
- 22. RNAi technology gene silencing zIn addition to the traditional strategies for vector construction and genetic modification strategies described, z RNAi (interference) technology is now becoming a new way to improve the contents and fight the diseases of crop plants. z The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. z In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the similar mRNA and degrades it. z In certain instances, the DNA expressing the target mRNA also undergoes methylation as a by-product of the degradation process
- 24. z The RNA-inducedsilencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double- stranded RNA (dsRNA) fragment, such as small interfering RNA (siRNA) or microRNA (miRNA).
- 25. Producing Human Antibodiesin Animals z Production of Monoclonal antibodies (Mabs) • Used to treat cancer, heart disease, and transplant rejection • HUMANIZED monoclonal antibodies were developed to prevent the human anti- mouse antibody (HAMA) response
- 26. A monoclonal antibody(mAb, more rarely called moAb) is an antibody produced from a cell lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. • Multiple myeloma. Multiple myeloma cells are abnormal plasma cells (a type of white blood cell) that build up in the bone marrow and form tumors in many bones of the body. Normal plasma cells make antibodies to help the body fight infection and disease. • A hybrid cell made in the laboratory by fusing a normal cell with a cancer cell, usually a myeloma or lymphoma, in order to combine desired features of each, as the ability of the cancer cell to multiply rapidly with the ability of the normal cell to dictate the production of a specific antibody.
- 27. Composition and function ofHAT medium HAT medium contains hypoxanthine-aminopterin-thymidine. Hypoxanthine, aminopterin, and thymidine selection (HAT) medium is commonly prepared from two stock solutions, 100× HT and 100× A. 1. To prepare 100 mL of 100× HT, dissolve 136 mg of hypoxanthine and 38 mg of thymidine in 100 mL of H2O suitable for tissue culture. This mechanism is also used in monoclonal antibody production. HAT(hypoxanthine–aminopterin–thymidine) medium is used for the selection of fused hybridoma cells in the process of monoclonal antibody production. After fusion of myelomas and spleen cells, HAT medium is used