Construction of gene-targeting vectors: A rapid Mu in vitro DNA transposition-based strategy generating null, potentially hypomorphic, and conditional alleles

Gene targeting into mammalian genomes by means of homologous recombination is a powerful technique for analyzing gene function through generation of t ransgenic animals. Hundreds of mouse strains carrying targeted alleles have already been created and recent modifications of the technology, in partic ular generation of conditional alleles, have extended the usefulness of the methodology for a variety of special purposes. Even though the standard pr otocols, including the construction of gene-targeting vector plasmids, are relatively straightforward, they typically involve time-consuming and labor ious gene mapping and/or sequencing steps. To produce various types of gene -targeting constructions rapidly and with minimum effort, we developed a st rategy, that utilizes a highly efficient in vitro transposition reaction of phage Mu, and tested it in a targeting of the mouse Kcc2 gene locus. A vas t number and different types of targeting constructions can be generated si multaneously with little or no prior sequence knowledge of the gene locus o f interest. This quick and efficient general strategy will facilitate easy generation of null, potentially hypomorphic, and conditional alleles. Espec ially useful it will be in the cases when effects of several exons within a given gene are to be studied, a task that necessarily will involve generat ion of multiple constructions. The strategy extends the use of diverse reco mbination reactions for advanced genome engineering and complements existin g recombination-based approaches for generation of gene-targeting construct ions.