Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice.
Gene trapping in murine embryonic stem cells is a proven method for the simultaneous identification and mutation of genes in the mouse. Gene trap vectors are designed to detect insertions within genes through the production of a fusion mRNA transcript, making the identification of the endogenous gene possible by 5' rapid amplification of cDNA ends (RACE). Although the amplification of specific cDNAs can be achieved rapidly, cloning and screening of informative-sized cDNAs has proven to be time consuming. To eliminate the need for cloning, we have developed a method for solid-phase sequencing of 5' RACE products. More than 150 independent gene trap cell lines were analyzed, and sequence information was obtained for every line successfully amplified by RACE. With the vector used in this study, 40% of the cell lines were found to contain properly spliced gene trap events. The remaining lines were either spliced inefficiently or contained deletions of the vector. These results highlight the advantage of sequencing gene trap integrations before further characterization. This work now paves the way for large-scale gene trap screens in mice and should greatly accelerate the functional analysis of the mammalian genome.
Townley
D J
DJ
Biotechnology and Biological Sciences Research Council (BBSRC), University of Edinburgh, UK.
Avery
B J
BJ
Rosen
B
B
Skarnes
W C
WC
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D018076:DNA, Complementary; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D017423:Sequence Analysis, RNA; D013234:Stem Cells
D018076:DNA, Complementary