Fig. 2. Generation of Ihh-deficient ES cells. A targeting construct designed to replace the first exon with a neoR cassette (A) was used to generate Ihh null cell lines (clone 14-61 and 14-63I (not shown)), identified using PCR (not shown) and Southern blot analysis (B). An NcoI digest yields an 8 kb endogenous allele and an 8.8 kb recombinant allele (see heterozygous clone14). Absence of Ihh mRNA expression and continued expression of Shh mRNA in Ihh null cell lines was demonstrated via RT-PCR analysis of embryoid body RNA (C) and loss of hedgehog response was detected by in situ hybridization analysis for Ptch in day 10 embryoid bodies (D).
