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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-NeoR 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.
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