Construction of Gene Targeting Vectors From Lambda KOS Genomic Libraries

Biotechniques. 1999 Jun;26(6):1150-6, 1158, 1160. doi: 10.2144/99266rr02.

Abstract

We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked 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.

MeSH terms

  • Animals
  • Bacteriophage lambda / genetics*
  • Cloning, Molecular / methods
  • Genetic Vectors*
  • Genomic Library*
  • Mice
  • Mutagenesis
  • Plasmids
  • Recombinant Proteins / genetics
  • Restriction Mapping
  • Transformation, Genetic
  • Yeasts / genetics

Substances

  • Recombinant Proteins