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Study of Gene Function by Gene Targeting and Down Regulation

Study of Gene Function by Gene Targeting and Down Regulation. Inje University YongBum Koo. Definition. Gene Targeting - Complete and permanent destruction of a gene - Alteration of gene function by replacing an original gene with a mutated allele.

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Study of Gene Function by Gene Targeting and Down Regulation

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  1. Study of Gene Function by Gene Targeting and Down Regulation Inje University YongBum Koo

  2. Definition • Gene Targeting - Complete and permanent destruction of a gene - Alteration of gene function by replacing an original gene with a mutated allele. - Systemic, tissue-specific or cell-specific • Gene knockdown - Permanent or temporary down-regulation of a gene - Systemic, parts of tissue or organ-specific

  3. Principles of Gene Targeting

  4. Embryonic Stem Cells

  5. Gene Targeting in ES cells 1. Electroporation of ES cells with a linearized targeting vector DNA 2. Selection -positive selection(Neo-r expression, antibiotic G418) -negative selection(TK expression, gancyclovir) 3. Expansion and DNA Prep 4. Targeting site analysis by sourthern analysis

  6. Southern Analysis

  7. Vector for Gene Targeting

  8. Site Specific Recombinase • Cre -bacteriophage P1 Cre recombinase - Interacts with loxP site • Flp - a site-specific recombinase encoded by the 2 micron plasmid of yeast - Interacts with FRT site

  9. Breeding chimera • ES cell : XY produce agouti color • C57BL/6 host cells : produce black color • Get chimera(Agouti coat color) • Cross male chimera with female C57BL/6 and get agouti color • Of these agouti colored mice, 50% will carry the targeted gene(hemizygous).

  10. Tail DNA analysis • Cross the hemizygous mice and get homozygous mutant mice • Tail DNA analysis • Trait observation-histology, physiology, genetics, biochemistry, structure, behavior

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