Fig. 1. Targeted deletion of the AF-1-containing A/B region of RXR. (a) Representation of the wild-type RXR1 isoform (WT RXR1) and the mutant RXR (RXRA/B). Exons E1 to E3 are shown. The functional domains are depicted as followed: AF-1, activation function 1; AF-2 AD, activation function 2 activating domain; DBD, DNA-binding domain (region C); LBD, ligand-binding domain (region E). The RXRaf1 mutation leads to the production of an RXR protein truncated from amino acid 11 to 132. BglII(Bg) and NheI(N) restriction sites were introduced to allow detection of the mutant allele. E[2-3] contains the first codon of exon 2 fused to the 3' part of exon 3. The deletion is represented by a black box. (b) The WT RXR locus, the targeting construct and the mutated loci obtained after replacement (I), and subsequent Cre-mediated excision of the ‘floxed’ TK-NEO cassette (II). The star represents the mutation E[2-3] described in (a). LoxP sites are represented by black arrowheads. Probes A, B and C are 0.7 kb ScaI-SpeI, 0.6 kb EcoRI-BglII and 0.3 kb BamHI-SpeI fragments, respectively. The size of the restriction fragment that allow identification of WT and targeted alleles (I) and (II) by Southern blot analysis using probes A, B, C and neo are indicated below (in kilobases). N, NheI; Bg, BglII; Sc, ScaI; S, SpeI; E, EcoRI; H, HindIII; B, BamHI; X, XbaI; Xh, XhoI. (c) Southern blotting of ES cell DNA digested with BglII or SpeI and hybridized with probe A, B and neo, as indicated. WT (+/+) and RXRaf1 mutant alleles before (+/(I)) or after (af1/+) excision of the ‘floxed’ TK-NEO cassette are indicated. (d) Detection of RXRA/B protein. Nuclear extracts prepared from 12.5-day-old embryos WT (+/+), af1 heterozygote (af1/+), af1 homozygote (af1o) and RXR-null mutants (-/-) (Kastner et al., 1994) were analyzed by western blotting with the anti-RXR monoclonal antibody 4RX3A2, directed against a C-terminal epitope.