biotech~2 hoza
TRANSCRIPT
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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
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Recombinant DNA Technology
• Site directed mutagenesis–Base pair substitution–Insertion–Deletion–Plasmid or PCR based approach
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GeneticsGenetics
Mutant phenotype
Mutant allele
DNA sequence
Protein sequence
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Reverse GeneticsReverse Genetics
Protein sequence
DNA sequence
Mutant allele
Mutant phenotype
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Recombinant DNA Technology
• Restriction fragment length polymorphism–Restriction digest–Gel or gel + Southern blot–Identify linkage to the mutant allele
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Recombinant DNA Technology
• Gene disruption–Gene knockout–Insert selectable marker into gene
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Recombinant DNA Technology
• Two-step expression vector–Chromosomal lac inducible T7 RNA pol–T7 RNA pol initiates expression of plasmid
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Recombinant DNA Technology
• Bacterial expression of hGH–No human signal sequence–Add bacterial signal sequence
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Recombinant DNA Technology
• Engineered bacteria–Synthesize new antibiotics–Actinorhodin (blue)–Medermycin (brown)–Hybrid mederrhodin A (reddish purple)
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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.
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Recombinant DNA Technology
• Yeast –Vectors–Transformation–Gene regulation
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Recombinant DNA Technology
• Genetic engineering in plants–Ti plasmid–Agrobacterium tumefaciens–Crown gall disease–Genetically engineered plants
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Recombinant DNA Technology
• Transgenic Drosophila–Bacterial beta galactosidase gene–P elements
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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
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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).
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Recombinant DNA Technology
• Rodent gene knockouts–ES cell knockouts
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Recombinant DNA Technology
• Transgenic rodents–Rat growth hormone responsive to heavy
metals
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Recombinant DNA Technology
• Transgenic fish–Hormone complex with strong promoter in
salmon
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Recombinant DNA Technology
• Gene Therapy–Injection of fertilized egg–Germinal therapy–Somatic therapy
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Recombinant DNA Technology
• Tracking disease alleles–Amniocentesis–Southern blotting–Common genetic disorders
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Genomics
• Overview• VNTRs • Linkage• RAPDs• FISH and painting• Genomes• Yeast two hybrid system• Microarrays
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Genomics
• VNTRs–Simple sequence repeats–Highly variable–Excellent for linkage and human identity
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Genomics
• Linkage–Allele/marker segregates with a disorder
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Genomics
• RAPDs–Highly variable–Unknown origin–Reproducibility?
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Genomics
• Chromosomal approaches–FISH–Paints–Study of large genomic alterations
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Genomics
• Somatic cell hybrids–Full complement of rodent chromosomes–One or more human chromosomes–Chromosome level PCR based mapping
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Genomics
• Genome sequences–Bacteria–Arabidopsis
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Genomics
• Yeast two hybrid system–Target and bait–Detect gene interaction
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Genomics
• Microarrays–High throughput –Genotyping –Gene expression
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