Transgenic organisms

this ppt explains about the transgenic organisms and their uses

1. Transgenic organisms M.Iyyadurai , I year M.Sc. Microbiology, Centre for marine science and technology, ( MS university) Rajakamangalam

2. Introduction An  organism whose genetic characteristics have been altered by the insertion of a  modified gene  or a gene  from another  organism using the  techniques  of genetic  engineering . The most common genetically modified (GM) organisms are crop plants Microbes are the first organisms to be genetically modified

3. Cont … The FDA Center for Veterinary Medicine(CVM) regulates genetically altered animal products Currently no transgenic animals have been approved for human consumption Transgenic animals have been approved for use as biopharm animals (for producing drugs and hormones) and they produce such products as milk and wool

4. Other terms that mean the same thing: Genetically engineered Genetically modified organisms Recombinant DNA ( rDNA ) technology

5. What is not a GMO? Does not include Mutants. Fusion of animal cells unless the product can form an animal. Plants formed x protoplast fusion. Plants formed by embryo rescue or invitro fertilisation or zygote implantation. Organisms formed by natural DNA transfer

6. General priniciples of transgenic organisms Transgenic organisms  contain foreign DNA that has been introduced using biotechnology.  Foreign DNA (the  transgene ) is defined here as DNA from another species, or else recombinant DNA from the same species that has been manipulated in the laboratory then reintroduced.  The process of creating transgenic organisms or cells to be come whole organisms with a permanent change to their germline has been called either  transformation  or  transfection .

7. Cont … To make a transgenic cell, DNA must first be transferred across the cell membrane, (and, if present, across the cell wall), without destroying the cell. Techniques for transfer the transgenic gene : Electroporation:   In some cases, naked DNA (meaning plasmid or linear DNA that is not bound to any type of  carrier) may be transferred into the cell by adding DNA to the medium and temporarily increasing the porosity of the membrane, for example by  electroporation. Microinjection: When working with larger cells, naked DNA can also be microinjected into a cell using a specialized needle. 

8. Cont … Vectors : Vectors for transformation/transfection include  vesicles made of lipids or other polymers that surround DNA; various types of particles that carry DNA on their surface; and infectious viruses and bacteria that naturally transfer their own DNA into a host cell, but which have been engineered to transfer any DNA molecule of interest . Different types of vectors: Plasmids ex: bacterial plasmid, cosmid , yeast plasmid. Viral vectors ex: retro virus, lenti virus, adeno virus, adeno - associated virus. Expression vector ex: pGEX – 3X

9. Cont … Techniques for know the presence of transgene in host: PCR Southern blotting Techniques for know the expression of transgene in host: Reverse-transcription PCR RNA blotting Western blotting (protein blotting)

10. History

11. Recombinant DNA technology : Recombinant DNA is the general name for taking a piece of one DNA, and  and combining it with another strand of DNA .   Recombinant DNA is also sometimes referred to as "chimera." By combining two or  more different strands of DNA, scientists are able to create a new strand of DNA .   The most common recombinant process involves combining the DNA of two  different organisms. 

12. Methodology:

13. Methods for transfer rDNA to host: There are three different methods by which Recombinant DNA is made . Transformation , Phage Introduction, Non-Bacterial Transformation.

14. Techniques for transfer the transgene into host : An organism can acquire a new trait by having a new gene introduced into its DNA. Under appropriate conditions, the new gene can be inserted into the DNA of a cell; this gene will be transcribed and translated into protein along with all the other genes being expressed in the cell. DNA microinjection Retro viral vectors Embryonic stem cell transfer Agrobacterium mediated transformation

15. DNA microinjection: The new gene is inserted directly into the fertilized ovum Most commonly used method Only 5% or less of the treated eggs become transgenic progeny Need to check mouse pups for DNA , RNA and protein ( by some specific assay method) Expression will vary in transgenic offspring: due to position effect and copy number

16. R etro virus mediated gene transfer: Retroviral vectors can be used to create transgenic animals

17. Embryonic stem cell transfer: Genetically engineeredembryonic stem (ES) cells can be used to create transgenic animals This method allow for gene targeting via homologous recombination .

18. The Nobel Prize in Physiology or Medicine, 2007 Mario R. Capecchi , Martin J. Evans and Oliver Smithies for their discoveries of "principles for introducing specific gene modifications in mice by the use of embryonic stem cells" M. Capecchi Sir M. Evans O . Smithies Univ. of Utah Cardiff Univ., UK UNC Chapel Hill

19. Agrobacterium mediated transformation: Agrobacterium mediated gene transfer  is one of the most widely used  methods of gene transfer .  Agrobacterium tumefaciens  is a bacterial plant pathogen found in the soil which causes crown gall disease in plants . The basic mechanism that works behind it is, transfer of a segment or small piece of bacterial DNA to the plant genome. This bacterial DNA segment gets incorporated in the plant genome, stabilizes there and transcribes or expresses itself.

20. Cont … The bacterial DNA causing disease is situated on a plasmid, Ti plasmid (Tumour inducing plasmid, 200kb) instead of on a bacterial chromosome. The segment which is actually transferred to plant genome is situated on T-DNA (transferred DNA, 23kb) of this plasmid.

21. Applications : Transgenic plants: Pest Resistance Crops Growing GM crops can help to reduce the use of chemical pesticides, e.g., Bt Cotton Tolerance GM crops have made more tolerant to abiotic stresses (cold, drought, salt, heat, etc .) Reduction in Post-harvest Losses They have helped to reduce post harvest losses, e.g., Flavr Sarv transgenic tomato . Prevention of Early Exhaustion of Fertility of Soil: Increased efficiency of min­eral usage by plants prevents early exhaustion of fertility of soil. Increasing Nutritional Value of Food: GM plants enhance nutritional value of food, e.g., golden rice is rich in vitamin A.

22. Cont … Herbicide Resistance: Herbicides (weed killers) do not harm the GM crops. Alternative Resources to Industries: GM plants have been used to create alter­native resources to industries in the form of starches, fuels and pharmaceuticals. Researchers are working to develop edible vaccines, edible antibodies and edible interferon’s Disease Resistance: Many viruses, bacteria and fungi cause plant diseases. Scien­tists are working to create genetically engineered plants having resistance to these diseases. Phytoremediation Plants such as popular trees have been genetically engineered to clean up heavy metal pollution from contaminated soil.

23. Cont … Transgenic microorganism: Crop Production and Protection: Several bacteria have been modified by introduc­tion of foreign genes to control, ( i ) insects by production of endotoxins, (ii) fungal disease by production of chitinases , which suppress fungal flora in the soil and (iii) by production of antibiotic which will degrade the toxin produced by pathogen. There are also positive measures where the N 2  fixing efficiency of bacteria Rhizobia can be increased by transfer of useful nif genes, nif means nitrogen fixation . Biodegradation of Xenobiotic and Toxic Wastes: Bacteria can be modified geneti­cally for degradation of xenobiotic and other waste material. Bacterial genes for this purpose are isolated from bacteria found at waste sites. Production of Chemicals and Fuels: Genetic engineering also has an important impact on microbial production of chemicals and fuels. Examples: ( i ) genetically engineered strains of Bacillus amyloliquefaciens and Lactobacillus casei have been prepared for pro­duction of amino acids on a large scale (ii) E. coli and Klebsiella planticola carrying genes from Z. mobilis could utilize glucose and xylose to give maximum yield of ethanol.

24. Cont … Living Factory for the Production of Proteins: In bacteria, genetic engineering turns the bacterium into a living factory for the production of proteins. Examples: The transfer of genes for human insulin , human growth hormone ( hGH ) and bovine growth hormone.

25. Cont … Transgenic animals Biological Products: Transgenic animals that produce useful biological products can be created by the introduction of the portion of DNA (or genes) which codes for a particular product such as human protein (a-1-antitrypsin) used to treat emphysema, tissue plasmogen activator (goat), blood clotting factors VIII and IX (sheep) and lactoferrin (cow ). In 1997, the first transgenic cow, Rosie, produced human protein- enriched milk (2.4 gms per litre). The milk contained the human alpha- lactalbumin . It is a more balanced product for human babies than natural cow-milk. Vaccine Safety: Transgenic mice are being formed for use in testing the safety of vaccines before they are used on human beings. Transgenic mice are being used to test the safety of the polio vaccine.

26. Chemical Safety Testing: It is called as toxicity/safety testing. Transgenic animals are developed that carry genes exposed to the toxic substance and their effects are studied. Normal Physiology and Development: Transgenic animals are specifically devel­oped to study how genes are regulated, and how they affect the normal functions of the body and its development, e.g., study of complex factors involved in growth such as insulin-like growth factor. Study of Diseases: Many transgenic animals are developed to increase our under­standing of how genes contribute to the development of disease so that investigation of new treatments for diseases is made possible. Now transgenic models exist for many human diseases such as cancer , cystic fibrosis, rheumatoid arthritis, Alzheimer’s disease, haemophilia, thalessaemia , etc . Growing of Spare Parts: Spare parts (e.g., heart, pancreas) of pig for human use can be grown through the formation of transgenic animals.

27. Risks associated with Genetic Modification 1. Safety Potential human health implications. Potential environmental impact. Out-crossing Inevitable out-crossing of transgenic plants with naturally occurring ones. Creation of super-weeds Creation of biological weapons. 2. Access and Intellectual Property Domination of world food production by a few companies and developing countries.

28. 3. Ethics “Playing God” Tampering with nature by mixing genes among species. 4. Labeling Not mandatory in some countries (e.g., Canada and the United States). Mixing GM crops with non-GM confounds labeling attempts. 5. Society New advances may be skewed to the interests of rich countries.

29. Biodiversity Addition of Bt gene into plants including corn, potatoes and cotton to increase resistance to plants Bt gene obtained from Bacillus thuringiensis (a soil bacterium that produces a natural insecticide) Problem: plants producing Bt toxin are releasing toxin in pollen Pollen from a Bt plant was dusted on to milkweed: - only 56% of young monarch butterfly larvae lived - whereas pollen from organic plants dusted on the milkweed produced a survival rate of 100%. Approximately half of the monarch butterfly population live in the “corn belt” of the USA = this new gene could have serious repercussions for this organism

30. Conclusion: Genetic engineering has emerged as a novel tool for overcoming incompatibility and species barriers between crops and their wild relatives and other organisms . It deals with specificity and high precision to ensure the identification of the exact gene of interest, isolate and insert it in the precise recipient organism and be certain that the progeny with required attributes has been generated . Genetic modification allows breeders to carry out not only single gene transfer but also transfer of clusters of genes from one organism to another.