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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- 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
- Permanent or temporary down-regulation of a gene
- Systemic, parts of tissue or organ-specific
1. Electroporation of ES cells with a linearized targeting vector DNA
-positive selection(Neo-r expression, antibiotic G418)
-negative selection(TK expression, gancyclovir)
3. Expansion and DNA Prep
4. Targeting site analysis by sourthern analysis
-bacteriophage P1 Cre recombinase
- Interacts with loxP site
- a site-specific recombinase encoded by the 2 micron plasmid of yeast
- Interacts with FRT site