A molecular strategy designed for the rapid screening of gene traps based on sequence identity and gene expression pattern in adult mice.
We have devised a strategy to rapidly screen gene traps in mouse embryonic stem (ES) cells based on DNA sequence information and an in vitro analysis of gene expression. After the initial identification of ES cell clones expressing beta-galactosidase, tagged RNA transcripts were immediately cloned and sequenced in order to determine their identities. Novel gene sequences found were used to probe northern blots to examine the expression patterns of their cognate genes. Our initial characterization of 30 cDNA clones indicated that more than half of the tagged sequences were novel mouse genes and of these 40% showed a restricted pattern of expression in adult mouse tissues. This molecular characterization of gene traps is quick, reliable and well suited for the large-scale screening of mammalian developmental genes. Furthermore, since gene trap insertion frequently disrupts the tagged host gene, the ES cells can be used to produce transgenic animals for a genetic analysis of gene function.
Holzschu
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Section of Genetics & Development, Cornell University, Ithaca, NY 14853, USA.
Lapierre
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Neubaum
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Mark
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D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004622:Embryo, Mammalian; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics; D007763:Lac Operon; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA; D013234:Stem Cells; D019714:Tissue Inhibitor of Metalloproteinases; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D018076:DNA, Complementary; D006023:Glycoproteins; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D001616:beta-Galactosidase