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S. Babu, B.E. Knox; Characterization of the Red Cone Opsin Promoter in Xenopus Laevis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3531.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To understand the transcriptional regulation underlying cell specific expression of the red cone opsin gene. Methods: A 1.8 kb 5’ upstream fragment was isolated from a Xenopus BAC library. Reporter constructs with the 5’ region driving the expression of either EGFP or luciferase were made and tested for function in transgenic Xenopus and in transient transfection assays. The effect of Otx and maf-family transcription factors on RCOP promoter was evaluated using transfections in non-retinal cells. Results: In transgenic tadpoles, expression was predominant in the eye, and in some animals with high expression also in pineal and hind brain. In tadpoles (stage ~46-66) and in froglets, expression from the transgenic promoter construct was found only in cones. In older animals (stages >52), expression was quite variable in intensity and exhibited mosaic patterns. Transient transfections of Xenopus embryos demonstrated head-specific expression, as expected for a cone-specific gene. Sequence analysis of the upstream region identified a TATA box at –24 position, a potential Crx-binding site, an NRE-like site and a site similar to the GC/GT boxes. To study the proteins that regulate this promoter, HEK293 cell transfections were done with several transcription factors present in the developing retina. XOtx5b/Crx and XL-maf/Nrl stimulated the promoter 5.5 fold and 4.5 fold respectively. Both XOtx5b/Crx and XL-maf/Nrl stimulated transcription in an additive manner (~9 fold). A number of other transcription factors involved in eye field specification and in maintenance of retinal function are being tested for their involvement in cone-specific expression. Conclusions and Future Direction: The immediate 5’ upstream of the red cone opsin of Xenopus has elements necessary for cell-specific expression in cones. Further dissection of the promoter is essential to understand the cis-elements which regulate the expression of the gene
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