The Cre-loxP recombination system has been widely used to assess gene function in vivo, particularly when combined with established gene targeting technology. While targeted mutations and conditional alleles are routinely generated in 129 Sv/Ev ES cell lines and analyzed in 129 B6 hybrid progeny, most Cre transgenic lines have been generated in a different genetic background. Therefore, experiments using Cre excision are often performed in a different genetic background than the original knockout, complicating interpretation of phenotypes. In addition, because of transgene positional effects, several Cre transgenic lines are usually required in order to obtain one with the desired pattern and level of Cre. To circumvent this we generated an X-linked Cre line in the 129S5/SvEvBrd (129S5) line from which the widely used AB series of embryonic stem (ES) cells were derived and many floxed alleles are available. A Cre expression cassette under the control of the human cytomegalovirus (CMV) promoter was inserted into an insertional targeting vector designed to target the X-linked hypoxanthine phosphoribosyl transferase (Hprt) locus (Zhang et al., 1994)(Fig. 1A). This locus was selected because mutations of Hprt in mice had been shown to have minimal affects (Dunnett et al., 1989). Moreover, targeting the Hprt locus provides a convenient negative selection for accurately targeted clones. The targeting vector was linearized within the region of homology, and upon targeted insertion this will duplicate exon 2 and exon 3, mutating the Hprt locus. Following electroporation into AB1 ES cells, G418-and 6-TG-resistant clones were selected and targeting was confirmed by Southern analysis (Fig. 1B). The targeted allele HprtTg (CMV-Cre) Brd was established in mice using routine procedures.

To assess Cre activity, male HprtTg (CMV-Cre) Brd (CMV-Cre) mice were mated to females carrying a floxed PGK-Neo targeted allele. Because the Cre transgene is X-linked, all female progeny carry the transgene (inherited from their father). Cre mediated excision of the floxed PGKNeo was scored by PCR amplification of the tail DNA (Fig. 2A). The recombined allele generates a product that is 2.3 kb smaller than that of the nonrecombined allele because of the excision of the PGKneo cassette. The CMV promoter provides strong and constitutive expression in many cell types (Schmidt et al., 1990). In