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Recombinant DNA Technology

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  1. Recombinant DNA Technology • Site directed mutagenesis • Genetics vs. Reverse Genetics • Gene expression in bacteria and viruses • Gene expression in yeast • Genetic engineering in plants • Genetic engineering in animals • Gene therapy in mammals • Detection of disease alleles

  2. Recombinant DNA Technology • Site directed mutagenesis • Base pair substitution • Insertion • Deletion • Plasmid or PCR based approach

  3. Genetics Mutant phenotype Mutant allele DNA sequence Protein sequence

  4. Reverse Genetics Protein sequence DNA sequence Mutant allele Mutant phenotype

  5. Recombinant DNA Technology • Restriction fragment length polymorphism • Restriction digest • Gel or gel + Southern blot • Identify linkage to the mutant allele

  6. Recombinant DNA Technology • Gene disruption • Gene knockout • Insert selectable marker into gene

  7. Recombinant DNA Technology • Two-step expression vector • Chromosomal lac inducible T7 RNA pol • T7 RNA pol initiates expression of plasmid

  8. Recombinant DNA Technology • Bacterial expression of hGH • No human signal sequence • Add bacterial signal sequence

  9. Recombinant DNA Technology • Engineered bacteria • Synthesize new antibiotics • Actinorhodin (blue) • Medermycin (brown) • Hybrid mederrhodin A (reddish purple)

  10. Recombinant DNA Technology • Baculovirus expression • 150 kb genome. • Insect cells. • Low frequency recombination inserts foreign gene into viral genome. • Recombinant virus do not have coat protein.

  11. Recombinant DNA Technology • Yeast • Vectors • Transformation • Gene regulation

  12. Recombinant DNA Technology • Genetic engineering in plants • Ti plasmid • Agrobacterium tumefaciens • Crown gall disease • Genetically engineered plants

  13. Recombinant DNA Technology • Transgenic Drosophila • Bacterial beta galactosidase gene • P elements

  14. Recombinant DNA Technology • Transgenic sheep • Beta lactobumin promoter • Active only in mammary tissue • Microinjection into ovum • Transplant and test by PCR • High levels of transgene expressed in milk

  15. Recombinant DNA Technology • Rodent gene knockouts • Create targeting vector • Homologous recombination in cells • 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).

  16. Recombinant DNA Technology • Rodent gene knockouts • ES cell knockouts

  17. Recombinant DNA Technology • Transgenic rodents • Rat growth hormone responsive to heavy metals