HoxB6-Cre transgenic mice express Cre recombinase in extra-embryonic mesoderm, in lateral plate and limb mesoderm and at the midbrain/hindbrain junction.

Conventional gene targeting, by homologous recombination in embryonic stem cells, usually results in the loss of function of the targeted gene in all cells and tissues, from the earliest time of expression (Muller, 1999). For genes that are required developmentally, complete loss of function may lead to an embryo lethal phenotype. If the gene is also expressed at later developmental stages or in the adult, early lethality will prevent analysis of later gene function. This limitation can be overcome by using the Cre/loxP recombination system in which the loss of gene function can be controlled both in a tissue and developmental stage specific manner (Lobe and Nagy, 1998; Marth, 1996). We have generated transgenic mice in which Cre recombinase has been placed under the regulation of the well-characterized limb/LPM enhancer element derived from the HoxB6 gene (see Fig. 1; Becker et al., 1996; Eid et al., 1993). We derived three HoxB6-Cre lines, two on the FVB/N background and one on the C57Bl/6NCr background. To determine whether the Cre transgene was expressed, HoxB6-Cre mice were crossed to mice carrying a floxed allele of the nodal gene, known to be able to undergo Cre-mediated deletion (unpublished). Analysis of tail biopsy DNA of F1 offspring of the FVB/N founder (HB6C33) and one of the C57Bl/6NCr founders (HB6A5) revealed Cre-mediated deletion of nodal (data not shown), proving that Cre is expressed in these transgenic mice. Subsequent analysis showed that F2 animals could inherit the deleted allele in the absence of the HoxB6-Cre transgene, indicating that Cre-mediated deletion of nodal occurs in the germ line. To determine whether Cre transgene expression during embryonic development followed the expected pattern for the HoxB6 enhancer, HB6C33 mice were crossed to floxed Rosa26 reporter mice. Both the R26R strain (Soriano, 1999) and the ROSA26flox strain (Mao et al., 1999) were used. E6.5 to E11 embryos were collected for whole mount X-gal staining. The earliest expression was detected at E7.5 in extraembryonic mesoderm (Fig. 2A and B). Continued expression in this region was seen in subsequent stages (Fig. 2C and D) and in the allantois from the earliest point of its appearance (Fig. 2D and E). The first embryo-specific expression was found in the posterior of E8/8.5 embryos in the extreme distal lateral plate mesoderm, predominantly in the splanchnopleure contiguous with the extra-embryonic yolk sac that also showed continued expression at this stage (Fig. 2F and G). Expression in the midbrain/hindbrain junction region was first detected at E8.5 and continued through all stages examined (Fig. 2H, I, N, and P). Sectioning showed that expression was within neural ectodermal cells (Fig. 2J and K for E9.5). At E9.5 Cre expression was detected throughout the posterior lateral plate mesoderm and also in intersomitic spaces (Fig. 2I, L, and M). At E10.5 and E11.5, expression was found in both the developing fore and hind limbs (Fig. 2N, O, and P). Expression in the forelimb did not extend to the most anterior aspect (Fig. 2O). A similar pattern was seen for the HB6A5 line (data not shown). Both HoxB6-Cre transgenic lines have been bred to homozygosity and will provide a useful tool for deleting floxed alleles specifically in the extra-embryonic tissues