Construction of gene targeting vectors from lambda KOS genomic libraries

We describe a highly redundant murine genomic library in a new lambda phage , lambda knockout shuttle (lambda KOS) that facilitates Be very rapid const ruction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40 000 i ndependent genomic clones The subpools are arrayed into a 96-well format th at allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-c opy number pKOS plasmids, wherein the genomic inserts are automatically fla nked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify-the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.