Campomelic dysplasia (CD), a semilethal human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in the SRY-related transcription factor gene SOX9 (Foster et al., 1994; Wagner et al., 1994). The mouse Sox9 gene, located on distal chromosome 11, is expressed during chondrogenesis (Wright et al., 1995), and is a key regulator of cartilage differentiation (Bi et al., 1999) and testis development (Vidal et al., 2001). Sox9 is expressed in a variety of embryonic tissues (Ng et al., 1997), suggesting further functions for Sox9/SOX9 during organogenesis. Indeed, CD patients show malformations in nonskeletal organs such as brain, heart, and kidneys (Houston et al., 1983; Mansour et al., 1995).

Recently, Sox9+/J mutant mice have been generated by a classical gene targeting approach and were shown to phenocopy the skeletal malformations observed in CD (Bi et al., 2001). However, because Sox9+/J mice die shortly after birth, it was not possible to generate a stable Sox9 mutant mouse line. To circumvent this problem and to provide a tool for an in-depth analysis of Sox9 functions in skeletal development and the formation of other organs, we have generated a conditional Sox9 mutant allele in mice using the Cre/loxP system (Nagy, 2000; Le and Sauer, 2001)(Fig. 1). Five correctly targeted embryonic stem (ES) cell clones were identified, and, following successful deletion of the neo cassette by transient in vitro expression of Cre recombinase (Taniguchi et al., 1998), two clones were microinjected into C57BL/6 blastocysts. Germline transmission was obtained for both clones and two Sox9-flox mouse lines have been established by backcrossing to C57BL/6 mice. Both Sox9flox/flox homozygous male and female mice are viable and fertile and do not show any obvious phenotype.