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W.S. Stark, K.G. Pineda, C.F. Thomas; Rhodopsin-Promoter - GFP (Green Fluorescent Protein)-Reporter Analysis in Drosophila . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2853.
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Purpose: We are developing a convenient method to determine what controls opsin expression. Methods: Photoreceptive organelles (rhabdomeres) were viewed in live Drosophila with the deep pseudopupil in fluorescence and confocal microscopes. Transgenic Drosophila in which promoters of opsins Rh1, Rh3 and Rh4 drive GFP were made by Franck Pichaud and and Claude Desplan at New York University. Results: Flies raised on our normal food, in which the chromophore precursors were yellow corn meal and beta carotene (0.125 mg/ml), show green fluorescence in rhabdomeres. GFP was also in cell bodies as ascertained by a blurring of the image relative to rhabdomeres viewed by transmitted light. Normal rhodopsin-metarhodopsin conversions showed that GFP did not interfere with photoreceptor function. Beta carotene (0.4 mg/ml) added to deprivational medium resulted in striking fluorescence. Fluorescence was variable but substantially decreased in vitamin A deprived flies. Yeast food, as well as retinoic acid (2.4 mg/ml) added to deprivational medium, are expected to activate opsin's promoter without providing chromophore precursor (Picking et al. Exp Eye Res 63, 493-500, 1996); they increased emission above the vitamin A deprived level, but the emission was very variable. Although docosahexanoic acid (DHA) was proposed to be a ligand for the retinoid X receptor (de Urquiza et al., Science 290, 2140-2144, 2000) we found that adding DHA (0.5 mg/ml) to deprivational medium barely increased fluorescence at all. Brain heart infusion (18.5 mg/ml) added to deprivational medium increased emission strikingly even though these flies had little or no opsin (Picking et al.). Conclusions: GFP expressing stocks and optical methods in live flies expedite promoter-reporter analysis of opsin gene transcription and photoreceptor function. Because of the variability, a reasonable number of flies must be sampled.
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