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DNA technology Ch. 10

DNA technology Ch. 10. Cloning genes Into bacteria Into plants Into animals. Recombinant bacteria. Ch 10 p. 281. Human Growth Hormone ( hGH ) cloned into bacteria (1980s). Pituitary dwarfism mutation in hGH gene hGH protein is produced by pituitary gland

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DNA technology Ch. 10

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  1. DNA technology Ch. 10 • Cloning genes • Into bacteria • Into plants • Into animals

  2. Recombinant bacteria • Ch 10 p. 281

  3. Human Growth Hormone (hGH) cloned into bacteria (1980s) • Pituitary dwarfism mutation in hGH gene • hGH protein is produced by pituitary gland • Pre-1985 hGH from cadaver brains Drawbacks? • Today • hGH cloned 26 inches tall

  4. Cloning steps: • 1. isolate hGH mRNA from normal human

  5. 2. reverse transcribe to cDNA • How is cDNA different from DNA?

  6. 3. ligate hGH cDNA into plasmid vector

  7. 4. transform bacteria  grow bacteria and they express hGH • 5. purify protein

  8. Other cloned drugs made by bacteria Human insulin for diabetics 1987 Factor VIII for hemophiliacs 1993 Interferon for chemotherapy 1993 EPO for anemia 1992 FSH for fertility clinics 1996 TPA to prevent blood clots 1996 Hepatitis B vaccine bGH to increase milk yield in cows

  9. Advantages of rbacteria/drugs • Clean • Worlds supply in one lab • Cheap ?

  10. When rBST injected into cows  digestive systems are more efficient at converting feed to milk. 8 gallons of milk/day/cow  9 • 120 studies have found no differences in milk from rBST-supplemented cows. National Institutes of Health, the Congressional Office of Technology Assessment and the drug-regulatory agencies of Britain, Canada and the European Union, Department of Health and Human Services • Traces of BST are found in milk from all cows, supplemented or not. rBST is broken down in the human gut, does not enter blood stream • Even if injected into the human bloodstream, has no biological activity. • What do you think?

  11. Affect of high human growth hormone in childhood • ….. Pituitary tumor

  12. Cloning into plants (GM) pg 282 • Transgenic plants • Plants acquire new genetic trait by direct introduction of gene • We have been modifying plant genes by breeding for 1000’s of years

  13. Getting a gene into a plant embryo A natural system: Agrobacterium tumefaciens bacteria infects plants  crown gall disease (tumor) at wound sites Agrobacteriumtumefaciens cells attached to a plant cell. From Genome News Network and Martha Hawes.

  14. 1. Engineer Ti plasmid • Remove tumor inducing genes • Add gene of interest

  15. 2. transform agrobacterium with Ti plasmid Ti plasmid is 200kb

  16. 3. wound plant and infect with agrobacterium

  17. 4. gene of interest transferred to plant cell ssT-DNA ~20kb excised • Infection stimulates excision of 30 kb region of Ti called T-DNA  insertion into chromosome

  18. 5. grow explant • = transgenic plant Agrobacterium transgenic plant Virigina Tech

  19. Why is this referred to as horizontal gene transfer? • How could this be used to introduce engineered genes into a plant cell?

  20. Last year, 12 million farmers in 23 countries grew 282.4 million acres (114.3 million hectares) of GM crops genetically modified foods in US do not require labeling to notify consumers.

  21. GM plants 2007

  22. genetically modified plants Fungus chitinase gene Resistance gene to potato beetle have been approved in the US and in Canada. Osmotin gene to tolerate salt stress Nicotine-free GM tobacco iron-storage gene from wild soybeans and vitamin E has been enhanced by the use of genes from rice and rock cress. 2008

  23. Benefits • Increased crop yield • Resistance to drought, freezing • Decreased use of pesticide • Decreased use of herbicides • Increased nutrition • Increased shelf life • Can remove allergens • Drawbacks increased seed costs pesticide resistant bugs new allergens harmful to insects? may spread to other plants

  24. Bacillus thuringiensis (Bt) produce a protein crystal toxic to insects. • The toxin genes are on a plasmid • The proteins bind to receptors in insect's intestine. Humans do not have these receptors…………………….

  25. Bt transgenic corn Ch. 10 pg. 284 • Corn engineered with gene that codes for a Bt toxin More on Bt corn Univ. MN

  26. Effect of insect infestation on cotton Non engineered cotton Bt cotton http://cls.casa.colostate.edu/TransgenicCrops/what.html

  27. Mustard plant genetically engineered to remove selenium from soil Can then grind and use in selenium deficient soils

  28. Golden Rice Oryza sativa 2012! • Vitamin A deficiency • Leading cause of childhood blindness (500,000 new cases /year)

  29. Engineer rice to produce genes needed for b carotene in endosperm • (phytoene synthase and phytoene desaturase) • Tissue specific expression cassette Promoter--- transit sequence--- gene--- - One serving of golden rice supplies enough beta-carotene to meet 10 percent of the daily requirement for  Vitamin A 

  30. Do we need legislation for labeling of GM foods? • Should GM genes, plants, animals, be patented?

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