A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.
We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.
Wurst
W
W
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Rossant
J
J
Prideaux
V
V
Kownacka
M
M
Joyner
A
A
Hill
D P
DP
Guillemot
F
F
Gasca
S
S
Cado
D
D
Auerbach
A
A
D000818:Animals; D001755:Blastocyst; D002678:Chimera; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D016254:Mutagenesis, Insertional; D009154:Mutation; D013234:Stem Cells; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase