Embryonic Stem Cells Part II - Embryonic stem cells Part II...

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Unformatted text preview: Embryonic stem cells Part II Applications Basic science linical Clinical Basic science applications of mouse ESC Knock out candidate genes, one at a time to mimic human disease or understand developmental processes (works only to some extent due to the fact that most disease are not single gene traits, but rather complex trait or polygenic) Over-express genes (same purpose) Gene trapping earch for gene expression profiles hat genes are involved Search for gene expression profiles - what genes are involved in stemness Use differentiation of ESC in vitro as model systems for development of specific organs(by-pass the need to work in animals) Knock out candidate genes Identify your gene of interest Construct your targeting vector Perform homologous recombination in ES cells Inject in blastocysts- obtain chimeras Breed for germline transmission Breed for homozygotes Knock out candidate genes onstructing your targeting vector/ESC Constructing your targeting vector/ESC mutant line Chimera Black coat into white coat Conditional knock-out strategy Heterozygous gene X floxed mouse Cre transgenic mouse x Exon 1 Exon 3 Exon 2 Cre recombinase Specific promoter loxP loxP Exon 1 Exon 3 Exon 2 Het for gene X/Cre mouse Homozygous floxed mouse (gene X fully expressed) Exon 3 Exon 1 x Exon 1 Exon 3 Exon 2 Cre recombinase Specific promoter Conditional knockout mouse Exon 3 Exon 1 Cre recombinase Gene is knocked out Exon 3 Exon 1 Specific promoter in the cells were Cre is expressed pithelial knock pithelial knock- ut mouse ut mouse Epidermis Epithelial knock Epithelial knock out mouse out mouse E1E2E3 E4 E5 E6 E7 loxPloxP Sebaceous gland Hair follicle Dermis Fat uscle Nerves re ediated Gene X flox (wild type) 14 Cre actin-Cre-ER+ moxifen Muscle Blood vessels Wild type skin Cre mediated DNA recombination tamoxifen E1E2E3 E5 E6 E7 loxP eratinocyte ecific All-cells GeneX 4 ene X 4 Keratinocyte specific Runx 4 knockout skin knockout skin Gene X (mutant) asic science applications of mouse ESC Basic science applications of mouse ESC Knock out candidate one gene at a time to mimic human disease or understand developmental processes (works only to some extent due to the fact that most disease are not single gene traits, but rather complex trait or polygenic phenomena) Over-express genes (same purpose) Gene trapping earch for gene expression profiles hat genes are involved in stemness Search for gene expression profiles - what genes are involved in stemness Use differentiation of ESC in vitro as model systems for development of specific organs(by-pass the need to work in animals) Classical transgenic approach Targeted transgenic approach Targeted transgenic into the HPRT ((hypoxanthine-guanine phosphoribosyltransferase) locus and ESC...
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Embryonic Stem Cells Part II - Embryonic stem cells Part II...

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