Fig. 6. Characterization of the novel, intestine-restricted homeobox gene Isx. (A) Northern analysis of whole mouse embryos at the indicated post-fertilization (E) days shows absence of Isx mRNA expression before E14. This feature distinguishes Isx from Cdx2, which is also expressed at peri-implantation stages. (B) In situ hybridization reveals epithelium-restricted expression of Isx transcripts in adult intestine; right panel shows results with a sense probe. (C) The Isx HD is most closely related to that found in the Paired family. Unrooted phylogenetic analysis using the CLUSTAL_W algorithm (20 of 28 Paired proteins are shown) reveals closest homology to Pax3, Pax7 and Prrx1. (D) Isx gene targeting strategy. 3.5 kb EcoRI (E) and 2.5 kb XbaI (X) genomic fragments were isolated from a 129/Sv BAC clone and used to flank a PGK-Neo^R cassette, positioned in reverse orientation, in the targeting construct. Positions of KpnI (K) sites enabled confirmation of gene targeting. (E) Correct targeting, with deletion of exon 1, was determined by Southern analysis of KpnI-digested DNA probed with the genomic fragments indicated in D. Homologous recombination produced the expected 12 kb and 4.5 kb bands with the 5' and 3' probes, respectively. (F) Northern blots confirmed loss of Isx expression after targeted gene disruption. RNA isolated from the cecum (Cec) and stomach (Sto) of nullizygous mutant (KO) mice and littermate controls (WT) was probed with Isx cDNA. Ethidium bromide staining of 28S RNA shows equal or excess loading of mutant samples.