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.
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
Recombinant DNA Technology • Site directed mutagenesis • Base pair substitution • Insertion • Deletion • Plasmid or PCR based approach
Genetics Mutant phenotype Mutant allele DNA sequence Protein sequence
Reverse Genetics Protein sequence DNA sequence Mutant allele Mutant phenotype
Recombinant DNA Technology • Restriction fragment length polymorphism • Restriction digest • Gel or gel + Southern blot • Identify linkage to the mutant allele
Recombinant DNA Technology • Gene disruption • Gene knockout • Insert selectable marker into gene
Recombinant DNA Technology • Two-step expression vector • Chromosomal lac inducible T7 RNA pol • T7 RNA pol initiates expression of plasmid
Recombinant DNA Technology • Bacterial expression of hGH • No human signal sequence • Add bacterial signal sequence
Recombinant DNA Technology • Engineered bacteria • Synthesize new antibiotics • Actinorhodin (blue) • Medermycin (brown) • Hybrid mederrhodin A (reddish purple)
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.
Recombinant DNA Technology • Yeast • Vectors • Transformation • Gene regulation
Recombinant DNA Technology • Genetic engineering in plants • Ti plasmid • Agrobacterium tumefaciens • Crown gall disease • Genetically engineered plants
Recombinant DNA Technology • Transgenic Drosophila • Bacterial beta galactosidase gene • P elements
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
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).
Recombinant DNA Technology • Rodent gene knockouts • ES cell knockouts
Recombinant DNA Technology • Transgenic rodents • Rat growth hormone responsive to heavy metals