Expression and promoter activation of the Rpe65 gene in retinal pigment epithelium cell lines

Purpose. To examine the expression and promoter activation of the retinal pigment epithelium (RPE)-preferentially expressed Rpe65 gene in the commonly available RPE cell lines. Methods. Reverse transcription coupled to polymerase chain reaction (RT-PCR) was performed after total RNA extraction from different RPE (ARPE-19, monkey, hTERT-RP1 and D407) and non-RPE (COS-7, HeLa, HepG2 and HS27) cell lines. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the 5' flanking region of the mouse Rpe65 gene. The involvement of a putative TATA box in the basal promoter expression was studied by site-directed mutagenesis in D407 cells and binding of TATA box-related transcription factors to that region was demonstrated by Electrophoretic Mobility Shift Assays (EMSA). Results. Expression of the human RPE65 cDNA was observed in all the RPE cell lines tested, and in COS-7 cells (monkey RPE65 cDNA). Transient transfections of the mouse Rpe65 promoter/luciferase transgene containing nested deletions of the Rpe65 5' flanking region showed that fragments containing bases -655 to +48 and -1240 to +48 generated specific promoter activity only in the D407 cell line. A promoter fragment from -49 to +48 directed basal promoter activity in all the cell lines tested. Part of this basal activity was due to a putative TATA box that specifically binds transcription factors contained in a D407 nuclear extract. Conclusion. Although transcription of the Rpe65 gene occurs in all the tested cell lines, we find that the D407 cell line is the only one capable of directing specific mouse Rpe65 promoter activity. This limits the study of the transcriptional regulation of the mouse Rpe65 gene in vitro to this particular cell line.