Conditional, floxed allele of the Krox20 gene.

Abstract

Krox20, initially identified as an immediate early serum response gene, encodes a transcription factor with a C2H2-type zinc finger DNA-binding domain (Chavrier et al., 1988, 1990). During mouse development, Krox20 is expressed in two rhombomeres (r3 and r5) of the segmented hindbrain and the neural crest derived from r5 (Schneider-Maunoury et al., 1993; Wilkinson et al., 1989), in immature and myelinating Schwann cells (Topilko et al., 1994), in hypertrophic chondrocytes and differentiating osteoblasts (Levi et al., 1996), and in hair follicles (Gambardella et al., 2000). In the adult, Krox20 expression has been shown to be maintained in myelinating Schwann cells and in the bones and has been observed in various other tissues, including specific neuronal populations of the cortex (Herdegen et al., 1993; Williams et al., 1995). Two Krox20 null mutant alleles, Krox20 and Krox20, have been previously generated by insertion of the E. coli lacZ gene and of the Cre recombinase gene, respectively, into the Krox20 locus (Schneider-Maunoury et al., 1993; Voiculescu et al., 2000). In homozygous mutants, hindbrain segments, r3 and r5, are eliminated (Schneider-Maunoury et al., 1993, 1997; Voiculescu et al., 2001), myelination is impaired in the peripheral nervous system (Topilko et al., 1994), and endochondral bone formation is affected (Levi et al., 1996). However, postnatal lethality prevented the analysis of late functions of Krox20. To investigate the role of Krox20 in the adult, we have generated a floxed allele (Sauer, 1998) that will be used to create conditional mutations. The second exon of the Krox20 gene encodes the largest part of the protein, including the DNA-binding domain. This exon was floxed by inserting a loxP site within the preceding intron and a floxed neoR cassette 3 of the gene (Fig. 1A). The targeting vector was electroporated into embryonic stem (ES) cells (CK35 line derived from the 129/SV Pasteur strain; Camus et al., 1996) and two out of 350 neo resistant ES cell clones were shown to contain a targeted allele (Krox20) by Southern blotting analysis (Fig. 1B). After injection of the recombinant ES cells into C57Bl6 blastocysts, mouse germ line transmission was obtained for one of them. The Krox20 line was established in a mixed C57Bl6/DBA2 background. As the neoR cassette could potentially interfere with Krox20 expression, Krox20 compound heterozygotes were generated. Indeed, analysis of lacZ expression in the hindbrain of 8.5 dpc embryos revealed that r3 is smaller in Krox20 embryos than in Krox20 embryos, whereas r5 is not affected (Fig.