title: Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles.
abstract: The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or 'recombineering', has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis. Here we applied Red/ET recombineering (using the lambda Redalpha/Redbeta recombinase pair) to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.
authors.affiliation: Biotec, Technische Universität Dresden, GmbH, c/o Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden, Germany 01307.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Testa
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kristina
authors.initials: K
authors.lastname: Vintersten
authors.forename: Vladimir
authors.initials: V
authors.lastname: Benes
authors.forename: W W M Pim
authors.initials: WW
authors.lastname: Pijnappel
authors.forename: Ian
authors.initials: I
authors.lastname: Chambers
authors.forename: Andrew J H
authors.initials: AJ
authors.lastname: Smith
authors.forename: Austin G
authors.initials: AG
authors.lastname: Smith
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
mesh_terms: D000483:Alleles; D000818:Animals; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D013234:Stem Cells / Q000502:physiology