title: Large-scale, saturating insertional mutagenesis of the mouse genome.
abstract: We describe the construction of a large-scale, orderly assembly of mutant ES cells, generated with retroviral insertions and having mutational coverage in >90% of mouse genes. We also describe a method for isolating ES cell clones with mutations in specific genes of interest from this library. This approach, which combines saturating random mutagenesis with targeted selection of mutations in the genes of interest, was successfully applied to the gene families of G protein-coupled receptors (GPCRs) and nuclear receptors. Mutant mouse strains in 60 different GPCRs were generated. Applicability of the technique for the GPCR genes, which on average represent fairly small targets for insertional mutagenesis, indicates the general utility of our approach for the rest of the genome. The method also allows for increased scale and automation for the large-scale production of mutant mice, which could substantially expedite the functional characterization of the mouse genome.
authors.affiliation: Omeros Corporation, 1420 Fifth Avenue, Suite 2600, Seattle, WA 98101, USA. sgragerov@omeros.com
authors.forename: Alexander
authors.initials: A
authors.lastname: Gragerov
authors.forename: Kyoji
authors.initials: K
authors.lastname: Horie
authors.forename: Maria
authors.initials: M
authors.lastname: Pavlova
authors.forename: Linda
authors.initials: L
authors.lastname: Madisen
authors.forename: Hongkui
authors.initials: H
authors.lastname: Zeng
authors.forename: Galina
authors.initials: G
authors.lastname: Gragerova
authors.forename: Alex
authors.initials: A
authors.lastname: Rhode
authors.forename: Io
authors.initials: I
authors.lastname: Dolka
authors.forename: Patricia
authors.initials: P
authors.lastname: Roth
authors.forename: Amanda
authors.initials: A
authors.lastname: Ebbert
authors.forename: Stephanie
authors.initials: S
authors.lastname: Moe
authors.forename: Christopher
authors.initials: C
authors.lastname: Navas
authors.forename: Eric
authors.initials: E
authors.lastname: Finn
authors.forename: John
authors.initials: J
authors.lastname: Bergmann
authors.forename: Demetrios K
authors.initials: DK
authors.lastname: Vassilatis
authors.forename: George N
authors.initials: GN
authors.lastname: Pavlakis
authors.forename: George A
authors.initials: GA
authors.lastname: Gaitanaris
mesh_terms: D000818:Animals; D053595:Embryonic Stem Cells; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome / Q000235:genetics*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D007041:Hypoxanthine Phosphoribosyltransferase