Dicistronic tagging of genes active in embryonic stem cells of mice

A gene-trap vector, pWH14, has been developed to tag genes expressed in embryonic stem (ES) cells of the mouse. The approach relies on the ability of the endogenous promoter to drive promoterless neo-IRES-lacZ construct producing a dicistronic mRNA consisting of the neomycin-resistance (neo) gene and the beta-galactosidase gene sequence. The neo gene produces a chimeric protein with the truncated product of the tagged gene and serves as a selectable marker for an insertion into an expressed gene. The internal ribosome entry site (IRES) sequence from murine encephalomyocarditis virus allows the translation of the second cistron, lacZ, to produce beta-galactosidase that can be used as a reporter for the expression of the tagged gene. The pWH14 vector was introduced into ES cells by electroporation, and the cells were selected for G418-resistance. About 50% of the G418-resistant colonies were stained positive for the beta-galactosidase activity. Southern analysis showed that each clone had one or more vector sequences integrated. Northern blot analysis of the clones positive for beta-galactosidase indicated that the fused RNAs containing the neo and the beta-gal genes were derived from the endogenous promoters of the tagged genes. Seven clones were chosen and injected into blastocysts, and chimeras were obtained. Two of the gene-trap insertions (wh14.1 and wh14.3) were transmitted through germ-line. In these two lines, the pattern of lacZ expression was restricted to early stages of embryos. This gene-trap vector may provide a means for tagging and studying the active genes in vivo in early embryogenesis.