Successful targeting of mouse Y chromosome genes using a site-directed insertion vector

Gene targeting via homologous recombination in mouse ES cells is now a routine method for addressing gene function in vivo. Several hundred genes mapping to all autosomes and the X chromosome have been mutated and analyzed in this way. In contrast, despite repeated attempts in several laboratories, including our own, there have been no reports of successful targeting of mouse Y chromosome genes. We show here that this problem can be overcome through the use of insertional targeting, rather than the usual replacement strategy. Using this method we have successfully targeted the mouse Y located Dby (dead box Y) and Eif2s3y (elongation initiation factor) genes. In addition, as Y chromosome genes are transcribed in ES cells, successful targeting and disruption of gene expression can be easily confirmed by RTPCR analysis of selected clones.