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Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings p.174 Biotechnology - use of living organisms to create products or help processes Ex. HGH, insulin Recombinant DNA - segment of DNA containing sequences from different organisms How is DNA manipulated? Restriction enzymes cut DNA at specific sites and create sticky ends G T G G T T A A C A C C A A T T A T T A C C T C C A G A G G A G G T C T G A T C G C T C T G T T C A G A A A C A C T G G C A A A G G T C T T A T GA GA T TC C C G G G T T C T T T C G A T C C A G AA C G G A A A T TC T C C TAG AG C A C G A G A C A A G C TA A G G T C T T G G C T T TA G Complementary ends will fuse to produce a long strand of DNA • The DNA is then integrated into the recipient cell’s chromosome Donated DNA Degraded DNA Crossovers Recipient cell’s chromosome Figure 12.1D Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Recombinant chromosome Plasmids are extra rings of DNA that replicate in bacteria. DNA can be inserted into plasmids. bacterium bacterial chromosome plasmid Cloning Vectors Bacterium Human cell Plasmid DNA Human protein Bacterial chromosome 1. Use restriction enzymes. 2. Insert gene into plasmid. recombinant DNA transformation 3. Transfer the plasmid back into bacterial cell. replication 4. Let bacterial cells replicate. bacterial clones Recombinant DNA products • “seed” protein for artificial snow • Insulin for diabetes treatment • Enzymes that clean up toxic waste spills • Growth Hormones (Human, Bovine) • TPA: Tissue Plasminogen Activator for treatment of heart attacks Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube Initial DNA segment • Selection of specific sequence 1 2 4 Number of DNA molecules Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 8 Figure 12.12 Gel electrophoresis sorts DNA molecules by size • Restriction fragments of DNA are compared by size Mixture of DNA molecules of different sizes Longer molecules Power source Gel Shorter molecules Glass plates Completed gel Figure 12.10 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings DNA forensics Egg microinjection to produce transgenic animal Credit: © Science VU/Visuals Unlimited Egg manipulation via microinjection. Grow bigger fish faster. Salmon with gene from another fish species • Uses of transformed animals: • Produce medicines • Study human diseases • Low-pollution pigs • Low-fat pork Figure 12.16 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Genetic engineering of plants Methods to insert DNA: 1. Ballistics 2. Protoplasts 3. Agrobacterium as vector - Ti plasmid Credit: © Brad Mogen An extremely large Agrobacterium tumefaciens tumor (crown gall disease) and secondary tumors on Kalanchoe stem. a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue). b The bacterium infects a plant cell and transfers the Ti plasmid. The plasmid DNA becomes integrated into a plant chromosome. c The plant cell divides. Its descendant cells may develop into a plant that can express the foreign gene. A young plant expressing a fluorescent gene product Fig. 11-12, p.171 Genetically modified crops • Golden rice with Vitamin A • Cotton resistant to boll weevil • Soybeans resistant to herbicide (Roundup) • Corn resistant to European corn borer • Rapeseed with healthier vegetable oil • Virus-resistant papaya Benefits and risks Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Gene therapy • Goal - Treat diseases caused by mutated genes • Method - Add a normal gene or block an abnormal gene in enough cells to restore normal function • Target - somatic cells Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Gene therapy may someday help treat a variety of diseases • treat disease by altering an afflicted individual’s genes Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid – Ex vivo (shown here) Retrovirus – In vivo 2 Infect bone marrow cell with virus – Stem cells 3 Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow 4 Inject cells Figure 12.19 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings into patient Which disorders are candidates for gene therapy treatment? • Disorders due to mutations in one or more genes • The responsible gene is known • The affected tissues are known and accessible Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Knockout gene therapy • Goal: turn off a gene that is causing a disorder • Strategies: – Antisense – Triple helix oligos – Spliceosome – Ribozyme Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Critical factors in choosing a vector Gene size - limited room in vector genome • Target tissue – What cells can the vector infect? • Integration into the genome – Without integration, only short-term effect – Random integration may disrupt other genes • Cell cycle stage (dividing vs. non-dividing) Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Photo courtesy of Van de Silva Gene Therapy Successes Ashanti de Silva successfully treated for ADA deficiency - 1990 Ryes Evans successfully treated for SCID - 2001 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Gene Therapy Problems Jesse Gelsinger died of complications due to an immune system response while participating in a clinical trial Three children treated for SCID developed leukemia due to disruption of a gene that regulates cell division Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Ethical and Social Issues • Patient safety while participating in clinical trials • Which applications are therapies and which are enhancements? – “Designer” babies • Access to gene therapies Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings