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

GeneticsGenetics

Mutant phenotype

Mutant allele

DNA sequence

Protein sequence

Reverse GeneticsReverse 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

Recombinant DNA Technology

• Transgenic fish–Hormone complex with strong promoter in

salmon

Recombinant DNA Technology

• Gene Therapy–Injection of fertilized egg–Germinal therapy–Somatic therapy

Recombinant DNA Technology

• Tracking disease alleles–Amniocentesis–Southern blotting–Common genetic disorders

Genomics

• Overview• VNTRs • Linkage• RAPDs• FISH and painting• Genomes• Yeast two hybrid system• Microarrays

Genomics

• VNTRs–Simple sequence repeats–Highly variable–Excellent for linkage and human identity

Genomics

• Linkage–Allele/marker segregates with a disorder

Genomics

• RAPDs–Highly variable–Unknown origin–Reproducibility?

Genomics

• Chromosomal approaches–FISH–Paints–Study of large genomic alterations

Genomics

• Somatic cell hybrids–Full complement of rodent chromosomes–One or more human chromosomes–Chromosome level PCR based mapping

Genomics

• Genome sequences–Bacteria–Arabidopsis

Genomics

• Yeast two hybrid system–Target and bait–Detect gene interaction

Genomics

• Microarrays–High throughput –Genotyping –Gene expression