Fig. 7. Pre-neoplastic cells do not express wild-type patched. (A) Primers used to distinguish wild-type and mutant patched transcripts. The predicted structures of wild-type and mutant patched transcripts are shown. In the mutant allele, a portion of exon 1 and all of exon 2 have been replaced with the ß-galactosidase coding sequence (lacZ). Thus, sequences within this region (detected by primers a and b) should only be present in wild-type transcripts. Sequences within exons 7 and 9 (detected by primers c and d) should be present in both wild-type and mutant transcripts. (B,C) Expression of wild-type and mutant patched. RNA from FACS-sorted GCPs, pre-neoplastic cells and tumor cells was subjected to real-time PCR analysis using primers c and d (exons 7-9, B) or primers a and b (exons 2-3, C). Expression levels were normalized to actin. Data represent means of three samples±s.e.m. Loss of wild-type patched expression was observed in six out of seven pre-neoplastic samples and 13 out of 13 tumor samples; see Fig. S1 in supplementary material for details.