Biotech~2 hoza

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Biotech~2 hoza

  1. 1. Recombinant DNA Technology <ul><li>Site directed mutagenesis </li></ul><ul><li>Genetics vs. Reverse Genetics </li></ul><ul><li>Gene expression in bacteria and viruses </li></ul><ul><li>Gene expression in yeast </li></ul><ul><li>Genetic engineering in plants </li></ul><ul><li>Genetic engineering in animals </li></ul><ul><li>Gene therapy in mammals </li></ul><ul><li>Detection of disease alleles </li></ul>
  2. 3. Recombinant DNA Technology <ul><li>Site directed mutagenesis </li></ul><ul><ul><li>Base pair substitution </li></ul></ul><ul><ul><li>Insertion </li></ul></ul><ul><ul><li>Deletion </li></ul></ul><ul><ul><li>Plasmid or PCR based approach </li></ul></ul>
  3. 9. Genetics Mutant phenotype Mutant allele DNA sequence Protein sequence
  4. 10. Reverse Genetics Protein sequence DNA sequence Mutant allele Mutant phenotype
  5. 11. Recombinant DNA Technology <ul><li>Restriction fragment length polymorphism </li></ul><ul><ul><li>Restriction digest </li></ul></ul><ul><ul><li>Gel or gel + Southern blot </li></ul></ul><ul><ul><li>Identify linkage to the mutant allele </li></ul></ul>
  6. 13. Recombinant DNA Technology <ul><li>Gene disruption </li></ul><ul><ul><li>Gene knockout </li></ul></ul><ul><ul><li>Insert selectable marker into gene </li></ul></ul>
  7. 15. Recombinant DNA Technology <ul><li>Two-step expression vector </li></ul><ul><ul><li>Chromosomal lac inducible T7 RNA pol </li></ul></ul><ul><ul><li>T7 RNA pol initiates expression of plasmid </li></ul></ul>
  8. 17. Recombinant DNA Technology <ul><li>Bacterial expression of hGH </li></ul><ul><ul><li>No human signal sequence </li></ul></ul><ul><ul><li>Add bacterial signal sequence </li></ul></ul>
  9. 21. Recombinant DNA Technology <ul><li>Engineered bacteria </li></ul><ul><ul><li>Synthesize new antibiotics </li></ul></ul><ul><ul><li>Actinorhodin (blue) </li></ul></ul><ul><ul><li>Medermycin (brown) </li></ul></ul><ul><ul><li>Hybrid mederrhodin A (reddish purple) </li></ul></ul>
  10. 25. Recombinant DNA Technology <ul><li>Baculovirus expression </li></ul><ul><ul><li>150 kb genome. </li></ul></ul><ul><ul><li>Insect cells. </li></ul></ul><ul><ul><li>Low frequency recombination inserts foreign gene into viral genome. </li></ul></ul><ul><ul><li>Recombinant virus do not have coat protein. </li></ul></ul>
  11. 28. Recombinant DNA Technology <ul><li>Yeast </li></ul><ul><ul><li>Vectors </li></ul></ul><ul><ul><li>Transformation </li></ul></ul><ul><ul><li>Gene regulation </li></ul></ul>
  12. 32. Recombinant DNA Technology <ul><li>Genetic engineering in plants </li></ul><ul><ul><li>Ti plasmid </li></ul></ul><ul><ul><li>Agrobacterium tumefaciens </li></ul></ul><ul><ul><li>Crown gall disease </li></ul></ul><ul><ul><li>Genetically engineered plants </li></ul></ul>
  13. 41. Recombinant DNA Technology <ul><li>Transgenic Drosophila </li></ul><ul><ul><li>Bacterial beta galactosidase gene </li></ul></ul><ul><ul><li>P elements </li></ul></ul>
  14. 43. Recombinant DNA Technology <ul><li>Transgenic sheep </li></ul><ul><ul><li>Beta lactobumin promoter </li></ul></ul><ul><ul><li>Active only in mammary tissue </li></ul></ul><ul><ul><li>Microinjection into ovum </li></ul></ul><ul><ul><li>Transplant and test by PCR </li></ul></ul><ul><ul><li>High levels of transgene expressed in milk </li></ul></ul>
  15. 45. Recombinant DNA Technology <ul><li>Rodent gene knockouts </li></ul><ul><ul><li>Create targeting vector </li></ul></ul><ul><ul><li>Homologous recombination in cells </li></ul></ul><ul><ul><li>Select for recombinants/knockouts G418 (Neo) eliminates wild type cells without vector integration and ganciclovir kills cells with randomly integrated vector (that carry the tk gene). </li></ul></ul>
  16. 47. Recombinant DNA Technology <ul><li>Rodent gene knockouts </li></ul><ul><ul><li>ES cell knockouts </li></ul></ul>
  17. 50. Recombinant DNA Technology <ul><li>Transgenic rodents </li></ul><ul><ul><li>Rat growth hormone responsive to heavy metals </li></ul></ul>
  18. 53. Recombinant DNA Technology <ul><li>Transgenic fish </li></ul><ul><ul><li>Hormone complex with strong promoter in salmon </li></ul></ul>
  19. 55. Recombinant DNA Technology <ul><li>Gene Therapy </li></ul><ul><ul><li>Injection of fertilized egg </li></ul></ul><ul><ul><li>Germinal therapy </li></ul></ul><ul><ul><li>Somatic therapy </li></ul></ul>
  20. 57. Recombinant DNA Technology <ul><li>Tracking disease alleles </li></ul><ul><ul><li>Amniocentesis </li></ul></ul><ul><ul><li>Southern blotting </li></ul></ul><ul><ul><li>Common genetic disorders </li></ul></ul>
  21. 61. Genomics <ul><li>Overview </li></ul><ul><li>VNTRs </li></ul><ul><li>Linkage </li></ul><ul><li>RAPDs </li></ul><ul><li>FISH and painting </li></ul><ul><li>Genomes </li></ul><ul><li>Yeast two hybrid system </li></ul><ul><li>Microarrays </li></ul>
  22. 64. Genomics <ul><li>VNTRs </li></ul><ul><ul><li>Simple sequence repeats </li></ul></ul><ul><ul><li>Highly variable </li></ul></ul><ul><ul><li>Excellent for linkage and human identity </li></ul></ul>
  23. 69. Genomics <ul><li>Linkage </li></ul><ul><ul><li>Allele/marker segregates with a disorder </li></ul></ul>
  24. 72. Genomics <ul><li>RAPDs </li></ul><ul><ul><li>Highly variable </li></ul></ul><ul><ul><li>Unknown origin </li></ul></ul><ul><ul><li>Reproducibility? </li></ul></ul>
  25. 75. Genomics <ul><li>Chromosomal approaches </li></ul><ul><ul><li>FISH </li></ul></ul><ul><ul><li>Paints </li></ul></ul><ul><ul><li>Study of large genomic alterations </li></ul></ul>
  26. 78. Genomics <ul><li>Somatic cell hybrids </li></ul><ul><ul><li>Full complement of rodent chromosomes </li></ul></ul><ul><ul><li>One or more human chromosomes </li></ul></ul><ul><ul><li>Chromosome level PCR based mapping </li></ul></ul>
  27. 80. Genomics <ul><li>Genome sequences </li></ul><ul><ul><li>Bacteria </li></ul></ul><ul><ul><li>Arabidopsis </li></ul></ul>
  28. 83. Genomics <ul><li>Yeast two hybrid system </li></ul><ul><ul><li>Target and bait </li></ul></ul><ul><ul><li>Detect gene interaction </li></ul></ul>
  29. 85. Genomics <ul><li>Microarrays </li></ul><ul><ul><li>High throughput </li></ul></ul><ul><ul><li>Genotyping </li></ul></ul><ul><ul><li>Gene expression </li></ul></ul>

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