Or introducing a mutation knock in by site directed

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or introducing a mutation (knock-in) by site-directed mutagenesis Molecular Biology of the Cell NBK26818/#A1654 How is it done?
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Generating a mouse model of Achondroplasia: First, the targeting vector is generated In tube (in vitro) Region of homology Region of homology
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If the construct inserts by Homologous Recombination, recombination will occur A) in the yellow (vector) regions only. B) in the blue and yellow regions only. C) in the red regions only. D) in all regions with equal probability. TK Neo r FGFR3 FGFR3 DNA construct Vector Vector Clicker question
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Gene targeting Step 2: Introducing the targeting vector into mouse embryonic stem cells Homologous recombination replaces the original gene with the mutated version Embryonic stem cells are isolated from an early mouse embryo
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Gene targeting, Step 3: selecting for ES cells in which the mutated gene replaced the original gene (and not incorporated randomly into the genome) Presence on neo gene confers resistance to antibiotic neomycin or its analog G418 Presence of tk (thymidine kinase) gene converts drug ganciclovir into a lethal derivative, so cells that have tk will die
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Ques%ons? text questions anytime during the class to: 858-360-4528
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Step 4: Selecting for ES cells in which homologous recombination occurred Molecular Cell Biology. 4th edition. Lodish H, et al. http:// books/NBK21632/#A1984
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You transfect ES cells from a brown mouse (dominant marker) with this targeting vector:
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