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JJ Janssen, AP M Janssen, AH M vanVugt, CA G G Driessen; Promoter Analysis of the Human RDH5 Gene . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3641.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify regulatory elements that are part of the promoter region of genes expressed specifically or highly in the retinal pigment epithelium (RPE). Methods: The human RDH5 gene encodes the retinoid cycle enzyme 11-cis retinol dehydrogenase. The highest levels of transcription of this gene are detected in the RPE. The human RDH5 promoter region was obtained by genomic PCR. From this originally 4 kb promoter fragment, shorter regions were subsequently cloned into the pGL3 vector in front of the luciferase reporter gene. For transfection studies the human RPE-derived cell lines ARPE19 and D407 were used. As control cell lines SK-mel, HELA, MDCK, HEK293 and COS7 were used. Electrophoretic mobility shift assays (EMSAs) were used to identify transcription factor binding sites within the RDH5 promoter. Results: RDH5 promoter constructs proved to be highly active in regulating luciferase reporter gene transcription compared to pGL3basic (negative control) and the positive control pGL2 containing the SV40-promoter and an SV40 enhancer. Of five control cell lines tested, the COS7 cell line was also found to be capable of supporting RDH5 promoter controlled luciferase gene transcription. EMSA analysis identified a transcription factor binding site downstream of the transcription initiation site. Removal of this sequence severely affects the RDH5 promoter activity. Conclusion: As for studies on the cellular retinal binding protein (CRALBP) and RPE65 promoter, D407 cells can be used to study the RDH5 gene promoter whereas ARPE19 cells cannot. A 100-bp (-50 to +50) RDH5 promoter fragment is sufficient to drive cell line specific low-level reporter gene transcription. For maximal promoter activity a 600-bp upstream region of the RDH5 gene suffices. Monkey kidney-derived COS7 cells and human RPE-derived D407 cells share the ability to support transcription of genes specifically or highly expressed in the RPE.
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