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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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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.

    - Systemic, tissue-specific or cell-specific

  • Gene knockdown

    - Permanent or temporary down-regulation of a gene

    - Systemic, parts of tissue or organ-specific


Principles of Gene Targeting


Embryonic Stem Cells


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


Southern Analysis


Vector for Gene Targeting


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


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).


  • 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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