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Construction of gene-targeting vectors by recombineering

Authors :
Song Choon Lee
Pentao Liu
Source :
Cold Spring Harbor protocols. 2009(9)
Publication Year :
2010

Abstract

INTRODUCTIONThe critical step in generating a knockout mouse is gene-targeting vector construction. Recombineering technology has greatly streamlined the vector construction process. This protocol describes a method for making conditional knockout (cko) targeting vectors using the pSim18 plasmid. This plasmid carries the three λ phage Red genes (Gam, Bet, and Exo) under the control of the pL promoter, which is in turn regulated by the temperature-sensitive CI857 repressor. Hence, the heat-inducible recombineering functions can be easily delivered to bacterial artificial chromosomes (BACs) using a simple plasmid transformation, allowing one to manipulate any cloned mouse genomic region in Escherichia coli for cko vector construction. The conditional targeting vectors described in this protocol generate a flexible reporter/null/conditional allele in the mouse.

Details

ISSN :
15596095
Volume :
2009
Issue :
9
Database :
OpenAIRE
Journal :
Cold Spring Harbor protocols
Accession number :
edsair.doi.dedup.....ae655961301843a31936fe163c9b7d0b