Fig. 1. Targeted inactivation of Pbx1 and gross morphology of wild-type and mutant embryos. (A) Schematic representation of the mouse Pbx1 cDNA, genomic locus, targeting vector and mutated allele following homologous recombination. In the Pbx1 cDNA, sequences derived from exon 3 and the homeodomain (HD) are shown as black and gray boxes, respectively. Approximately 20 kb of the Pbx1 locus flanking exon 3 (black box) are depicted along with mapped restriction sites. The targeting construct carries a PGK-neo cassette, inserted into the unique NheI site of Pbx1 exon 3, and the HSV-tk gene, shown as solid white boxes. The transcriptional orientations of the Pbx1 arms of homology are opposite to that of the PGK-neo cassette. The 5' and 3' external probes used for Southern blot analyses are shown as solid black boxes below the mutated allele. Restriction enzyme sites: E, EcoRI; N, NheI; S, SspI; X, XhoI. XmaI and NotI sites are in the targeting vector; the SspI site (S) was introduced to facilitate diagnostic analysis. (B,C) Southern blot analysis of Pbx1 alleles. DNA from cell lines (B) or mouse tissues (C) was analyzed with probes and enzymes indicated beneath the panels. Wild-type (16 and 6.5 kb) and mutant (7.2 and 5.5 kb) Pbx1 alleles for XmaI and SspI digests, respectively, are indicated to the right of each panel. TL, ES cell line used for Pbx1 gene targeting; clones 38 and 176, targeted ES cells that passed the Pbx1 mutation through the germ line; MEFs, mouse embryonic fibroblasts. (D) Western blot analysis of Pbx1b expression. Protein extracts of E16 embryos were subjected to western blot analysis using a monoclonal antibody specific for the Pbx1b isoform. Genotypes determined by Southern blotting are listed at the top. Right lane contains in vitro translated Pbx1b. Migrations and sizes (kDa) of molecular mass standards are indicated to the right. (E,F) Gross morphology of wild-type and Pbx1^-/- embryos at E13.5 (E) and E16 (F). Mutant embryos display massive subcutaneous edema (yellow arrowhead), pallor, slender thorax and abdomen, hypoplastic pinna (red arrowhead) and atypical hunched posture with abnormal orientation of both hind- and forelimbs (black arrowheads).