Lecture5genetargeting

Molecular biology of the cell garland science 2008 es

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Figure 22-91 Molecular Biology of the Cell (© Garland Science 2008) ES cells with gene knockout or knock-in Step 5: Introducing the ES cells with the mutant gene into a wild-type embryo Step 6: Implanting the embryo into “pseudopregnant” mouse http://www.ncbi.nlm.nih.gov/ books/NBK26818/figure/A1650/? report=objectonly
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Ques%ons?
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Problems with genes that act in multiple tissues, at multiple times How to l activate z the mutation at a time or in the tissue of choice? Solution: “conditional” knock-out, using microbial recombinase systems: Cre recombinase, recognises loxP sequences (originally found in P1 bacteriophage) FLP recombinase, recognizes FRTsequences (from yeast)
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Tissue-specific disruption of a gene using Cre/ LoxP system l floxed z gene “deleter” mouse, expresses cre recombinase: - Depending on the promoter to which Cre is fused, it can be expressed either in all cells, or just in a subset of cells (brain, liver, etc) For more information: http://cre.jax.org/introduction.html
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Tissue-specific disruption of a gene using Cre/LoxP system
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At home: summarize the differences between knockout, knock-in, and conditional knockout Knockout Knock-in Conditional knockout Purpose Position of neoR (intron? exon?) Position of regions of homology Position of TK Position of loxP
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In Achondroplasia paper, the researchers flanked (“floxed”) neo gene with loxP sites In tube In animal In animal Cre recombinase deleted neo gene
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Generating a mouse model of Achondroplasia: First, the targeting vector is generated In tube In ES cells In animal
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Summary
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Molecular Biology of the Cell Garland Science 2008 ES cells...

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