Dt40 gene disruptions: a how-to for the design and the construction of targeting vectors

Abstract

Genome projects have provided comprehensive gene catalogs and locus maps for many model organisms. Although sequence comparison and protein domain searches may suggest evolutionary conserved gene functions, genetic systems are still needed to determine the role of genes within living cells. Due to high ratios of targeted to random integration of transfected DNA constructs, the chicken B cell line DT40 has been widely used as a model for gene function analysis by gene knockout. Targeting vectors need to be carefully designed to introduce defined mutations and to ensure high targeting rates. In this review we summarize general guidelines for the design of targeting vectors which can be used for single, multiple or conditional gene knockouts, as well as site-directed genome mutagenesis in DT40.