title: A conditional knockout resource for the genome-wide study of mouse gene function.
abstract: Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. skarnes@sanger.ac.uk
authors.forename: William C
authors.initials: WC
authors.lastname: Skarnes
authors.forename: Barry
authors.initials: B
authors.lastname: Rosen
authors.forename: Anthony P
authors.initials: AP
authors.lastname: West
authors.forename: Manousos
authors.initials: M
authors.lastname: Koutsourakis
authors.forename: Wendy
authors.initials: W
authors.lastname: Bushell
authors.forename: Vivek
authors.initials: V
authors.lastname: Iyer
authors.forename: Alejandro O
authors.initials: AO
authors.lastname: Mujica
authors.forename: Mark
authors.initials: M
authors.lastname: Thomas
authors.forename: Jennifer
authors.initials: J
authors.lastname: Harrow
authors.forename: Tony
authors.initials: T
authors.lastname: Cox
authors.forename: David
authors.initials: D
authors.lastname: Jackson
authors.forename: Jessica
authors.initials: J
authors.lastname: Severin
authors.forename: Patrick
authors.initials: P
authors.lastname: Biggs
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.forename: Michael
authors.initials: M
authors.lastname: Nefedov
authors.forename: Pieter J
authors.initials: PJ
authors.lastname: de Jong
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
authors.forename: Allan
authors.initials: A
authors.lastname: Bradley
mesh_terms: D000483:Alleles; D000818:Animals; D019295:Computational Biology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D005796:Genes / Q000235:genetics*; D005804:Genes, Lethal / Q000235:genetics; D056726:Genetic Association Studies / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome / Q000235:genetics*; D023281:Genomics; D005838:Genotype; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods; D010641:Phenotype; D016133:Polymerase Chain Reaction; D051381:Rats