Fig. 3

Detection of expression and identification of sequences of EYS gene transcripts corresponding to exon 42–43 that includes c.8805C > A in photoreceptor-directed fibroblasts from EYS-RP patients. a RT-PCR analysis of expression of EYS gene using primer pairs corresponding to exon 42–43. Expression levels of the EYS gene were clearly down-regulated by CRX, RAX, NeuroD and OTX2 transduction in Pt#1, Pt#2 and N#2 10 days after transduction. Y79 was used as a positive control. Interestingly, the exon 42–43 region of the EYS gene was expressed in human dermal fibroblasts without photoreceptor-induction. The expression level of the exon 42–43 fragment in default state fibroblasts was higher than in photoreceptor-directed fibroblasts. b Electropherogram corresponding to c.8805C > A by sequencing of RT-PCR products. The nucleotide sequence in this panel shows a complementary strand. The transcripts having c.8805C > A mutation were found both in photoreceptor-directed fibroblasts and default state fibroblasts from Pt #2. The peak amplitudes of normal and mutated bases on the electropherogram were nearly the same. c Analysis of expression levels of the EYS gene corresponding to exon 42–43. Vertical axis indicates relative expression (mean ± SD, n = 4 for Pt#1 and Pt#2, n = 3 for N#1, N#2 and N#3). The expression levels of the exon 42–43 fragment in default state fibroblasts were higher than in photoreceptor-directed fibroblasts 10 days after transduction. d Analysis of expression of the EYS gene in the default state fibroblasts by endpoint RT-PCR corresponding to exon 36–41. RT-PCR products amplified with the forward primer designed at exon 36 (EYS 7140) were detected only in Y79. On the other hand, RT-PCR products amplified with the forward primer designed at exon 37 (EYS 7286) were detected both in normal fibroblasts and Y79. These results suggest that the transcription start site of fibroblast-type exists between EYS 7140 (exon 36) and EYS 7286 (exon 37). 8093F and 9096R correspond to the forward and reverse primers used for the exon 42–43 fragments