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M. Haeri, P. D. Calvert, E. N. Pugh, Jr., B. E. Knox; Birefringence Bands in Rod Photoreceptors Originate from Diurnal Variation in Rhodopsin Packing into Outer Segment Disc Membranes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2727.
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To understand rhodopsin synthesis and transport during light/dark cycles and to understand the origin of birefringence banding pattern originally described by Kaplan, (Kaplan, Vis. Res. 24:1163-8, 1984).
Plasmids containing rod photoreceptor specific promoters driving the expression of rhodopsin fused to EGFP were constructed and used to make transgenic tadpoles by REMI. Transgenic frogs were maintained in a normal 12 hr light, 12 hr dark cycle prior to experiment upon which the durations of the light/dark cycles were altered for several weeks. The expression profiles of the transgene in outer segment compartments were then recorded from retinal explants using live cell confocal microscopy.
Rhodopsin-EGFP fluorescence appeared in a striped pattern perpendicular to and along the length of the rod outer segment. Animals raised in a 12/12 light/dark cycle generated a pattern that was in phase with the birefringence banding pattern. Rearing animals in complete darkness or with continuous light resulted in more uniform appearance of the fluorescence and in disappearance of the birefringence banding. It appears that discs made in the dark phase possess higher fluorescence density compared to those made in light phase. RTPCR examination of the message levels of the rhodopsin showed no variation with dark/light phase. Exchanging the Xenopus opsin promoter with promoters for transducin alpha or arrestin did not alter the banding pattern of rhodopsin-EGFP; however, other peripheral or intrinsic membrane proteins tagged with EGFP did not show the banding pattern.
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