Biotech~2 hoza

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


• 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


• Restriction fragment length polymorphism–Restriction digest–Gel or gel + Southern blot–Identify linkage to the mutant allele


• Gene disruption–Gene knockout–Insert selectable marker into gene


• Two-step expression vector–Chromosomal lac inducible T7 RNA pol–T7 RNA pol initiates expression of plasmid


• Bacterial expression of hGH–No human signal sequence–Add bacterial signal sequence


• Engineered bacteria–Synthesize new antibiotics–Actinorhodin (blue)–Medermycin (brown)–Hybrid mederrhodin A (reddish purple)


• Baculovirus expression–150 kb genome.–Insect cells.–Low frequency recombination inserts foreign

gene into viral genome.–Recombinant virus do not have coat protein.


• Yeast –Vectors–Transformation–Gene regulation


• Genetic engineering in plants–Ti plasmid–Agrobacterium tumefaciens–Crown gall disease–Genetically engineered plants


• Transgenic Drosophila–Bacterial beta galactosidase gene–P elements


• 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


• 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).


• Rodent gene knockouts–ES cell knockouts


• Transgenic rodents–Rat growth hormone responsive to heavy

metals


• Transgenic fish–Hormone complex with strong promoter in

salmon


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


• 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

Biotech~2 hoza

Technology

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