A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells.
Cultured mouse or human embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Chemical or insertional mutagenesis of Blm-deficient mouse ES cells can be used to generate genome-wide libraries of homozygous mutant ES cells, which are the substrates for conducting phenotype-driven loss-of-function genetic screens. However, the existing insertional mutation libraries are limited by incomplete genomic coverage. In this study, we have explored the use of piggyBac (PB) transposon-mediated mutagenesis to extend the genomic coverage of mutation libraries in Blm-deficient ES cells. A library composed of 14,000 individual gene-trap clones was generated and a recessive genetic screen conducted to identify cells with defects in DNA mismatch repair (MMR) genes. Independent mutations in all known genes of the pathway Msh2, Msh6, Pms2, and Mlh1 were recovered in these screens. The genomic coverage in this library confirms its utility as a new genetic resource for conducting recessive genetic screens in mammalian cells.
Wang
Wei
W
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.
Bradley
Allan
A
Huang
Yue
Y
D000818:Animals; D053843:DNA Mismatch Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D015698:Genomic Library; D051379:Mice; D018345:Mice, Knockout; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics*; D053484:RecQ Helicases / Q000502:physiology*
D004251:DNA Transposable Elements; C097151:Bloom syndrome protein; D053484:RecQ Helicases; D045643:DNA Repair Enzymes