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

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

9. Genetics Mutant phenotype Mutant allele DNA sequence Protein sequence

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

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

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

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

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

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

25. 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.

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

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

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

43. 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

45. 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).

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

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