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Advanced Genetic Manipulation

This text provides in-depth insights into the stages of developing transgenic organisms through genetic manipulation. It covers purpose identification, gene selection, development methods, DNA extraction, insertion processes, field testing, and approval requirements. The text discusses challenges and solutions in creating transgenic animals and plants, along with the importance of proper gene registration and testing for efficacy. Furthermore, it delves into the controversial aspects of patenting genes in the biotechnology field.

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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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