Fig. 1. Disruption of Hmx2 by homologous recombination, mRNA analysis and genotyping of embryos carrying an Hmx2^lacZ mutant allele. (A) Hmx2 wild-type locus, targeting construct and Hmx2 mutant alleles. Homologous recombination of the targeting construct into the Hmx2 locus results in the insertion of ires.lacZ.neo into the Hmx2 homeobox. Owing to the presence of translation stop codons in all three open reading frames in the ires.lacZ.neo cassette, this mutant allele produces ß-galactosidase and a nonfunctional truncated Hmx2 protein. The black boxes show the positions of the two Hmx2 exons. The homeobox is located in the second exon. The transcriptional orientation of Hmx2 is shown by an arrow. The positions and lengths of the probes used in Southern blot analysis and RNAse protection are also indicated by small black rectangles. (B) Southern blot assay from yolk sac DNA of wild-type, Hmx2^lacZ+/– and Hmx2^lacZ–/– embryos. Probe1 and Probe 2 are located outside of the targeting construct. Probe 1 detects a 17.0 kb wild-type NotI+ClaI fragment, as well as a 12.5 kb mutant NotI+ClaI band. Probe 2 hybridizes with a 5.7 kb mutant XbaI fragment instead of a 6.7 kb wild-type XbaI band because of the introduction of an additional XbaI by the ires.lacZ.neo cassette. Note: the XbaI site 5' to the Hmx2 gene is methylated in dam+ bacterial hosts. (C) RNAse protection analysis of Hmx2 RNA expression in wild-type, Hmx2^lacZ+/– and Hmx2^lacZ–/– embryos. A 242 genomic fragment spanning the Hmx2 homeobox was amplified by PCR using primers TL245 and TL246, and used as a template to prepare an antisense RNA riboprobe. RNA transcripts produced by the wild-type allele protect a fragment of 242 bp, whereas the Hmx2 mutant transcripts protect two fragments of 175 bp and 67 bp.