Large-scale, saturating insertional mutagenesis of the mouse genome.
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.
Gragerov
Alexander
A
Omeros Corporation, 1420 Fifth Avenue, Suite 2600, Seattle, WA 98101, USA. sgragerov@omeros.com
Horie
Kyoji
K
Pavlova
Maria
M
Madisen
Linda
L
Zeng
Hongkui
H
Gragerova
Galina
G
Rhode
Alex
A
Dolka
Io
I
Roth
Patricia
P
Ebbert
Amanda
A
Moe
Stephanie
S
Navas
Christopher
C
Finn
Eric
E
Bergmann
John
J
Vassilatis
Demetrios K
DK
Pavlakis
George N
GN
Gaitanaris
George A
GA
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
D043562:Receptors, G-Protein-Coupled; D007041:Hypoxanthine Phosphoribosyltransferase