A conditional floxed (loxP‐flanked) allele for the retinoic acid receptor beta (RARβ) gene

Retinoic acid (RA) acts through binding to nuclear receptors (RAR/RXR heterodimers), which belong to the ligand-inducible transcription factors superfamily (Chambon, 1996). To identify the functions of the three RAR isotypes ( , , and ) in the retinoid signaling pathway in vivo, their genes have been disrupted. Mice lacking one RAR isotype display only some of the defects resulting from postnatal vitamin A deficiency (VAD), whereas abnormalities recapitulating the fetal VAD syndrome were found in compound null mutants for two RAR isotypes. These findings led to the conclusion that (i) RARs are involved at multiple stages during morphogenesis and organogenesis of most tissues and (ii) in many instances functional redundancy allow, possibly artefactually, the remaining RARs to compensate for the missing one. All the compound null mutants die at birth or earlier during embryonic development, precluding analysis of RAR functions at later stages (Kastner et al., 1995, 1997; Mascrez et al., 1998; Mark et al., 1999; references therein). Thus, most of the physiological functions of RARs during development and in adults remain unknown. Additionally, germ-line mutations are inadequate to discriminate between cell-autonomous and non-cell-autonomous functions of RARs. Their elucidation will require spatio-temporally controlled somatic mutations. In this regard, the conditional mutagenesis approach based on the use of tamoxifen-inducible CreER recombinases should permit the inactivation of the RAR genes at any given time and in a chosen cell type (Metzger and Chambon, 2001). To this end, we report the generation of a mouse line harbouring a “floxed” (loxP-flanked) conditional allele at the RAR locus. The targeting vector encompasses exons 8–11 (E8– E11, amino-acid residues 204–448; Zelent et al., 1989) and contains a loxP-flanked neomycin selector gene upstream of exon 8, as well as a loxP downstream of exon 10 (Fig. 1A). After homologous recombination in embryonic stem (ES) cells, six positive clones (out of 278) containing a loxP-flanked L3 RAR allele were identified by Southern blotting (Fig. 1B). One of these clones (NE75) was electroporated with a pSG5Cre-encoding plasmid (J.M. Garnier and P.C., unpublished data). Cremediated recombination was analyzed in 86 subclones. In some ES cells, the entire loxP-flanked DNA fragment was excised (RAR L allele), whereas in 6 cases the neomycin cassette only was excised (floxed L2 allele, e.g., subclone NE75.4; Fig. 1C). Chimeric males derived from subclone NE75.4 transmitted the RAR L2 allele through their germ line, yielding the floxed RAR L2/L2 mouse line. Mice carrying the floxed L2 alleles expressed normal levels of RAR and were indistinguishable from wild-type littermates (Fig. 2B, C, and data not shown). To check whether Cre-mediated excision at the RAR locus can occur in vivo, RAR L2/L2 mice were crossed with CMV-Cre transgenic mice that express the Cre recombinase in germ cells (Dupé et al., 1997). PCR analysis of tail DNA revealed a Cre-dependent RAR L2 allele excision, yielding RAR /L heterozygous animals (Fig. 2A). The excised L allele bears a deletion of exons 9 and 10 that encodes most of the ligand-binding domain (amino-acid residues 263–384; Zelent et al., 1989) and causes a frameshift mutation. To verify that a null allele was generated, RAR /L mice were intercrossed to produce RAR L / homozygotes whose striatum did not express the RAR protein (Fig. 2B). Eye sections from RAR L / adult mice revealed the presence within the vitreous body of an abnormal retrolenticular mass of pigmented tissue (R, compare Fig. 2C with 2D), which exhibited a large base adherent to the lens (L). This retrolenticular membrane, caused by the persistence and hyperplasia of the primary vitreous body, characterizes RAR null mutants (Ghyselinck et al., 1997), showing