Recombinant DNA Technology

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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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Genetics
Mutant phenotype
Mutant allele
DNA sequence
Protein sequence
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Reverse 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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