Construction of Gene-Targeting Vectors by Recombineering

The 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 γ 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.