Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step. Recombineering: a powerful new tool for mouse functional genomics. Simplified generation of targeting constructs using ET recombination. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. An efficient recombination system for chromosome engineering in Escherichia coli. An improved recombineering approach by adding RecA to lambda Red recombination. A highly efficient recombineering-based method for generating conditional knockout mutations. DNA cloning by homologous recombination in Escherichia coli. A new logic for DNA engineering using recombination in Escherichia coli. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae.

A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. New method for generating deletions and gene replacements in Escherichia coli. Hamilton, C.M., Aldea, M., Washburn, B.K., Babitzke, P. Initial sequencing and comparative analysis of the mouse genome. A novel method for constructing gene-targeting vectors. The European dimension for the mouse genome mutagenesis program. International Mouse Knockout Consortium, Collins, F.S., Rossant, J. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Establishment in culture of pluripotential cells from mouse embryos. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Site-directed, mutagenesis by gene targeting in mouse embryo-derived stem cells. This protocol needs ∼2 weeks to construct a targeting vector, and several vectors can be easily handled simultaneously using common laboratory setup. To demonstrate the usefulness of this protocol, we report targeted disruptions of members of the cadherin gene family, focusing on those that have not been previously studied at the molecular genetic level. Finally, through a simple in vitro Gateway recombination, the modified genomic fragment is switched into a vector that contains negative selection cassettes, as well as unique sites for linearization.

Second, the vector is modified by recombineering to include a positive selection gene neo, from a variety of modular reagents. First step is the use of Red-pathway-mediated recombination (recombineering) to capture a genomic fragment into a Gateway-compatible vector.

This procedure combines various highly efficient recombination-based cloning methods in bacteria, consisting of three steps. We describe here a streamlined procedure for targeting vector construction, which often is a limiting factor for gene targeting (knockout) technology.

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