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Functional Analysis of Genes
Forward Genetics: Gene associated with mutant phenotype must be identified Reverse Genetics: Generation of mutant phenotype after isolating gene Targeted gene disruption (transgenic analysis) Mis-expression Ectopic expression Over expression Dominant inhibition
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Transgenic Analysis Random insertion of transgenes (for mutagenesis)
Targeted insertion of transgenes Knockout Knockin Requires special vectors contains flanking sequences to permit homologous recombination between construct and chromosome Contains selectable marker to permit survival only of homologous recombination and not non-homologous
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Cloning of Gene of Interest
Cut DNA with restriction enzyme Gene Ligate DNA fragment into vector with ligase enzyme HSV-tk Vector for homologous recombination
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Vector for Homologous Recombination
Gene 5’ flank 3’ flank HSV-tk
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Replace Gene with Selectable Marker
Transgene construct neor = neomycin phosphotransferase Such enzymes confer resistance to aminoglycoside antibiotics neomycin G418 kanamycin neor 5’ flank 3’ flank HSV-tk Vector for homologous recombination neo
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Transgenic Analysis Homologous Recombination
Homologous Chromosomes Gene 1A Ge Gene 1B neor 5’ flank 3’ flank HSV-tk homologous recombination vector Homologous recombination replaces one allele of the gene with the transgene construct Transgene construct
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Transgenic Analysis Homologous Recombination
Gene neor Homologous recombination replaces region of gene with neomycin resistance gene and disrupts generation of functional protein. Neor allows for cells to be selected for using antibiotic neomycin or G418. G418 neo
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Transgenic Analysis Non-Homologous Recombination
HSV-tk neor Non-homologous recombination inserts HSV thymidine kinase (HSVtk). The presence of this gene allows cells containing it to be killed by the thymidine analog gancyclovir or FIAU. The phosphorylated analog inhibits DNA synthesis. Only HSVtk will phosphorylate the nucleotide analog so only the cells with HSVtk will be killed – i.e. only non-homologous recombinants will die – homologous recombinants will live
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Transgenic Analysis Nucleotide Analogs
iodo fluoro arabinose
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Transgenic Analysis Introduction of Transgene into Cells
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Transgenic Analysis Recombination in ES Cell Culture
Electroporation, Transfection, or Microinjection & FIAU insensitivity
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Transgenic Analysis Genotype of transgenic ES cells:
bw/bw; BMP7+/- Genotype of host embryo cells: w/w; BMP7+/+ Genotype of chimeras Host derived cells: ES derived cells:
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Transgenic Analysis
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New Strategies - Floxing
Cre/Lox Cre recombinase from phage P1 LoxP site - 34bp site composed of 13bp inverted repeats Construct transgenic organisms with LoxP sites flanking target sequences
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Phage Integration/ Recombination
Figure 17.13B 15
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Cre-Lox Recombination
Figure 17.13A 16
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Cre-Lox Recombination
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Ways to Manipulate Floxed Genes
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