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Advanced Genetic Manipulation

Advanced Genetic Manipulation. Biotechnology 2. Creating a Transgenic or GM Organism. Stage I- Purpose Stage II- Development Stage III- Testing and Approval. Stage I- Purpose. First step of scientific method is to identify the problem.

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Advanced Genetic Manipulation

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  1. Advanced Genetic Manipulation Biotechnology 2

  2. Creating a Transgenic or GM Organism • Stage I- Purpose • Stage II- Development • Stage III- Testing and Approval

  3. Stage I- Purpose • First step of scientific method is to identify the problem. • Although the SM is followed the overall goal is usually profit. • Problems may come from individual or team brainstorming or as a result of previous research. • Risks, Expenses, and Time required are weighed against the potential importance (profits?)

  4. Selecting Genes • Genome Mapping is underway for many species • Lets researchers know where to find useful gene segments • In theory any gene from any organism can be removed • Genes from known allergens avoided for GM foods • Allergens may or may not be tied to gene sequences so scientists have to be careful when choosing

  5. Issues and Problems w/ TGO’s • Transgenic animals are more difficult and more expensive to create than TG plants • TG animal creation usually requires the use of specialized reproductive cells • Germ cells, stem cells, embryos, or haploid cells • Far fewer animal cells survive the process • 1-4% will develop into a full-term new born animal • Stem cells have great potential for cloning and genetic manipulation but production of new lines can only be obtained through the destruction of developing embryos

  6. Stage II- Development • Find target organism that will be changed • Locate, Isolate, Extract beneficial gene sequence • Usually hardest and longest part • Insert isolated gene into target organism • Biolistics is easiest, cheapest for plants • Micromanipulation for animals • Electroporation and Contact Absorption also common • All methods result in destruction of most cells

  7. DNA Extraction • Done with restriction enzymes and (radioactive) markers • Enzymes “read” DNA segment and cleave (cut) it after certain sequences • Markers indicate size and / or location of sequence • Have a known molecular weight so when analyzed w/ Gel Electrophoresis gene can be identified

  8. DNA Insertion- Vectors • Vectors attach the gene sequence to their own DNA and then insert into DNA of target organism • Bacteriophages (viruses)- inject DNA into cells • Bacteria (Agrobacterium tumefaciens) cause tumors and result in DNA changes to affected cells • Plasmids- Self-replication DNA loops inside protein coats of virus

  9. Figure 18.5 The lysogenic and lytic reproductive cycles of phage , a temperate phage

  10. 0.5 m T4 bacteriophage infecting an E. coli cell

  11. Review the steps of gene transfer

  12. Vectors • Can be inserted into organism several ways • Into physical wounds • Placed into contact w/ exposed cells • Placed into contact w/ dermal tissue • Some plants (Arabidopis) can be transformed via exposure to a liquid solution containing vectors • Usually a floral dip where developing flowers are literally dipped into the solution

  13. DNA Insertion- Fun Stuff • Electroporation • DNA sequence is placed close to cells in a solution that is then shocked to merge genetic info • Micromanipulation • Cell walls (plants)must be removed (also for electroporation) • Biolistics • Air or gun powder used to fire gold coated projectiles coated with target DNA into mass of plant cells • Most effective method for plant cells

  14. Micromanipulation • A tiny sharp syringe is used under a microscope to inject DNA through the cell membrane • Most TG animals done this way • Most exact method, fewest “casualty” cells • Either via ennucleation and replacement or by simply placing gene sequence into target cell(s) and pronucleus

  15. Stage II- Development • Test “new” organism for gene expression • Marker Genes used • Very useful in plants (black light bioluminescence) • Markers may not transmit to offspring b/c gene sequence of target gene and marker are not linked

  16. Stage III- Testing and Approval • Test efficacy of organism at addressing problem • Original DNA can impact new gene sequence • Determine if traits will transmit to future generations • Best to cross transgenic w/ natural • Backcrossing 2nd or 3rd generations might be needed when dealing w/ issues of complex heredity

  17. Field Testing • Organisms are kept separate in controlled environments isolated from natural populations until trait transmission is studied • Field testing ensures the inserted gene will not cause dangerous unintended consequences • Also that they perform the intended function

  18. Stage III- Testing and Approval • Gene and “new” organism registered w/ proper federal agency (who have overseen entire process anyway) • USDA • FDA • APHIS

  19. Patenting Genes • Very controversial since profits and expenses can be high • Disagreement often based on benefits and methods used to create and extract/insert the gene

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