Transgenics
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This document discusses the production of transgenic animals and plants. It describes three main methods for producing transgenic animals: DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. It also discusses 11 methods for transforming plants, including Agrobacterium-mediated transformation, biolistic transformation, and floral dip transformation. Finally, it lists some beneficial traits that have been engineered in transgenic plants, such as stress tolerance, herbicide tolerance, and increased nutritional quality.
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Transgenics
- 1. Presented By: Ms. Smita Shukla Assistant Professor
- 3. Plant Breeding vs. GMOs
- 5. Transgenic Animals A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology
- 9. How are transgenic animals produced?
- 11. Three basic methods of producing transgenic animals 1. DNA microinjection 2. Retrovirus-mediated gene transfer 3. Embryonic stem cell-mediated gene transfer
- 16. • Electroporation is a physical transfection method that uses an electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass into cells. • It is a highly efficient strategy for the introduction of foreign nucleic acids into many cell types, including bacteria and mammalian cells.
- 19. Life cycle of Retrovirus
- 20. Four classes of viral vector • Retrovirus- commonly used ( Offspring’s derived from this method are chimeric; not all cell carry the retrovirus i.e diverse genetic constitution) • Adenovirus • Herpesvirus • Adenoassociated Virus(AAV) Vectors
- 21. 3. Embryonic Stem Cell- Mediated Gene Transfer
- 22. • Embryonic stem cells • Formation of gametocytes • Injection into blastocysts • Injection into foster mother • Formation of new individual
- 23. How do transgenic animals contribute to human welfare?
- 24. The benefits of these animals to human welfare can be grouped into areas: • Agriculture • Medicine • Industry
- 25. 1. Agricultural Applications a) Breeding b) Quality c) Disease Resistance 2. Medical Applications a) Xenotransplantation b) human gene therapy c) nutritional supplements and pharmaceuticals
- 27. What are the ethical concerns surrounding transgenesis?
- 28. These ethical issues include questions such as: • Should there be universal protocols for transgenesis? • Should such protocols demand that only the most promising research be permitted?
- 29. • Is human welfare the only consideration? What about the welfare of other life forms? • Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than, or before, using live animals to alleviate animal suffering? • Should patents be allowed on transgenic animals, which may hamper the free exchange of scientific research?
- 31. Progress is being made on several fronts to introduce new traits into plants using recombinant DNA technology.
- 32. Making transgenic plants • Construction of a vector (genetic vehicle) Transfer methods in plants are: • Vector-mediated or indirect gene transfer • Vectorless or direct gene transfer
- 33. Vector-mediated or indirect gene transfer • Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used.
- 35. Vectorless or direct gene transfer • Foreign gene of interest is delivered into the host plant cell without the help of a vector. • Methods used for direct gene transfer in plants are:
- 36. 1. Chemical mediated gene transfer Chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells.
- 37. 2. Microinjection: Using fine tipped (0.5 - 1.0 micrometer diameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
- 39. 3.Electroporation: The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
- 42. 5.Transformation : Method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The uptake of plasmid DNA by E. Coli is carried out in ice cold CaCl2 (0-50°C) followed by heat shock treatment at 37- 45°C for about 90 sec.
- 43. 6. Conjugation: Natural microbial recombination process
- 44. 7. Liposome mediated gene transfer or Lipofection: • Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids
- 45. 8. Silicon carbide fibers mediated gene transfer in plants: Plant materials( cells in suspension culture,embroys,embryo derived callus etc..) is introduced into a buffer containing DNA & the silicon fibers which is then vortexed.
- 46. 9. Gene transfer using Pollen/ Pollen tube:
- 47. 10. Calcium Phosphate precipitation method for gene transfer
- 48. 11. Incubation of dry seeds, embryos, tissues or cells in DNA
- 49. 12. Sonication
- 50. Selection of transformed cells from untransformed cells
- 52. 1. Stress tolerance 2. Abiotic stress tolerance 3. Herbicide tolerance 4. Insect resistance 5. Phytoremediation 6. Disease Resistance 7. Soil Conservation 8. Delayed fruit ripening 9. Improvement of seed protein quality 10. Increased yield 11. Improved Nutritional quality 12. Edible vaccine
Editor's Notes
- A genetically modified organism (GMO) is an organism or microorganism whose genetic material has been altered to contain a segment of DNA from another organism. Modern recombinant DNA technology enables the “stitching together” of pieces of DNA, regardless of the source of the pieces. Since the 1980s, this technology has been used extensively in the lab by researchers for countless purposes: to make copies of genes or proteins, to determine gene function, to study gene expression patterns, and to create models for human disease. One application has been to generate food crops that are modified in a way that is advantageous to either the producer or the consumer. Currently the GM crops on the market have bacterial genes introduced into their genomes that encode for pest or herbicide resistance. In theory, this should cut down on the amount of chemicals a farmer needs to spray, but in practice that goal has not been realized as pests and weeds become resistant to the chemicals being used.
- Since the discovery of the molecular structure of DNA by Watson and Crick in 1953, molecular biology research has gained momentum. Scientists can now produce transgenic animals because, since Watson and Crick's discovery, there have been breakthroughs in: 1. Recombinant DNA (artificially-produced DNA) 2. Genetic cloning 3. Analysis of gene expression (the process by which a gene gives rise to a Protein) 4. Genomic mapping
- Gene transfer by microinjection is the predominant method used to produce transgenic farm animals. Since the insertion of DNA results in a random process, transgenic animals are mated to ensure that their offspring acquire the desired transgene.
- The mouse was the first animal to undergo successful gene transfer using DNA microinjection.6 This method involves: transfer of a desired gene construct (of a single gene or a combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell . The manipulated cell, which first must be cultured in vitro (in a lab, not in a live animal) to develop to a specific embryonic phase, is then transferred to the recipient female.
- The Desired gene construct is injected in the pronucleus of a reproductive cell using a glass needle around 0.5 to 5 micrometers in diameter. The manipulated cell is cultured in vitro to develop to a specific embryonic phase, is then transferred to a recipient female.
- A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. This method involves: Retroviruses used as vectors to transfer genetic material into the host cell, resulting in a 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
- This method involves: Isolation of totipotent stem cells (stem cells that can develop into any type of specialized cell) from embryos. The desired gene is inserted into these cells. Cells containing the desired DNA are incorporated into the host’s embryo, resulting in a chimeric animal. Unlike the other two methods, which require live transgenic offspring to test for the presence of the desired transgene, this method allows testing for transgenes at the cell stage.
- Breeding: Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate).Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, it offers the farmer an easy way to increase yields. Quality: Transgenic cows exist that produce more milk or milk with less lactose or cholesterol, pigs and cattle that have more meat on them, and sheep that grow more wool. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product. Disease resistance Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals. 2. Medical Applications xenotransplantation Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the United Kingdom alone. Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein. b) nutritional supplements and pharmaceuticals Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of diseases such as phenylketonuria (PKU), hereditary emphysema etc.. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs.4,21,23 Rosie’s milk contains the human gene alpha-lactalbumin C) Human gene therapy Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals could play a role.
- 3. Industrial Applications In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings. Toxicity-sensitive transgenic animals have been produced for chemical safety testing. Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions.
- Thoughtful ethical decision-making cannot be ignored by the biotechnology industry, scientists, policy-makers, and the public.
- CaCl2 breaks the cell wall at certain regions and binds the DNA to the cell surface
- It is a natural microbial recombination process and is used as a method for gene transfer. In conjuction, two live bacteria come together and the single stranded DNA is transferred via cytoplasmic bridges from the donor bacteria to the recipient bacteria.
- Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells.
- The fibers then penetrate the cell wall and plasma membrane, allowing the DNA to gain access inside the cells.