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Fig. 1. Targeted disruption of the Sufu gene. (A) The Sufu gene
consists of 12 exons and contains a putative PEST sequence. The carboxy
terminus is required for Gli binding
(Kogerman et al., 1999;
Dunaeva et al., 2003). In the targeting vector (middle), the intron 6-exon 7
junction and most of exon 7 was replaced with the neo gene. The
fragment used as an external probe in Southern blotting for screening and
genotyping is indicated. (B) Southern blot analysis of genomic DNA from 9.5
dpc wild-type, heterozygous and homozygous embryos. DNA was digested with
BamHI and hybridized with an intron 8 probe that detects the 9 kb
normal allele and the 11 kb targeted allele. (C) Northern blot analysis of 9.5
dpc total whole embryo RNA using a full-length Sufu cDNA probe
detected a 5.5 kb transcript in all genotypes, but aberrant 8.5 kb and 1.5 kb
transcripts were detected in the homozygous mutant. A 3' probe did not
detect any transcript in the homozygous mutant, but the expected 5.5 kb
transcript was detected in wild-type and heterozygous embryos. Gapdh
transcript is equal in all genotypes. (D) Western blot analysis of 9.5 dpc
whole embryo lysates using an anti-Sufu antibody that detects the carboxy
terminus. No Sufu protein expression was detected in the homozygous mutant,
but the expected 54 kDa band was present in wild-type and heterozygous
embryos. ß-Actin expression was equal in all genotypes.
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