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 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.
Wattler
S
S
Lexicon Genetics Incorporated, The Woodlands, TX, USA.
Kelly
M
M
Nehls
M
M
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors; D015698:Genomic Library; D051379:Mice; D016296:Mutagenesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics
D011994:Recombinant Proteins