A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome.
A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration "hot spots." Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.
Hansen
Jens
J
Institute of Developmental Genetics, GSF-National Research Center for Environment and Health, D-85764 Neuherberg, Germany.
Floss
Thomas
T
Van Sloun
Petra
P
Füchtbauer
Ernst-Martin
EM
Vauti
Franz
F
Arnold
Hans-Hennig
HH
Schnütgen
Frank
F
Wurst
Wolfgang
W
von Melchner
Harald
H
Ruiz
Patricia
P
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D006801:Humans; D051379:Mice / Q000235:genetics*; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional / Q000379:methods*; D010641:Phenotype; D016324:Sequence Tagged Sites; D013234:Stem Cells
D004247:DNA