Gene trap: An approach to rapidly identify, clone and mutate genes important in mouse development

I have developed the "gene trap" approach to screen for genes important for normal mouse development and physiology. A promoterless lacZ reporter construct containing a splice acceptor site was used to randomly generate lacZ fusions with endogenous genes in mouse embryonic stem (ES) cells. In principle, each gene trap insertion that synthesizes a lacZ fusion product places the reporter gene under the control of cis-acting regulatory sequences of the endogenous gene and creates a mutation in the endogenous gene by disrupting its normal coding potential.;ES cell lines containing gene trap insertions were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. The expression patterns of the lacZ reporter and the associated endogenous gene were compared in situ at three embryonic stages. The distribution of the endogenous transcript matched the sites of ;To demonstrate that the gene trap insertion disrupts endogenous gene transcription, RNA from mice homozygous for two gene trap insertions was analyzed. In these samples, the amount of normal endogenous transcripts, which would have indicated splicing out of the insertion, was negligible. Three gene trap insertions were transmitted to the germline and abnormalities were observed in mice homozygous for two of the three insertions.;These results prove the feasibility of the gene trap approach to identify genes based on patterns of gene expression, to generate insertional mutations that can result phenotypic defects, and to clone the interrupted endogenous gene.;A portion of the endogenous gene associated with gene trap integrations can be cloned directly from the lacZ fusion transcript. From three ES cell lines, cDNA was prepared from the lacZ fusion transcript and amplified by the polymerase chain reaction to recover endogenous sequences upstream of the gene trap splice acceptor. Sequence analysis of these clones and larger clones isolated from a conventional cDNA library showed that the splice acceptor was used properly. A single open reading frame was found in each clone that was in-frame with the lacZ coding sequences.