1 / 12

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.

britany
Download Presentation

Study of Gene Function by Gene Targeting and Down Regulation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related