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VJ Kefalov, Y Fu, N Marsh-Armstrong, KW Yau; Functional Expression of Human Red Cone Opsin in Xenopus Laevis Rods . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1834.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Despite the similarities between rod and cone phototransduction cascades, the origin of the differences in properties between rods and cones is not well understood. In order to study the role that the visual pigment plays in determining the physiology of photoreceptors, we produced transgenic Xenopus expressing human red cone opsin in the rods. Methods: We made transgenic Xenopus frogs expressing human red cone opsin under the control of the CMV promoter. Cone opsin expression in the outer segment of rods was confirmed immunohistochemically. Using a suction electrode, membrane current was recorded from single rod photoreceptors from transgenic and wild-type animals. Results: Rods expressing human red cone opsin showed normal size, dark current, and time to peak of the dim-flash response. The single-photon response was reduced by 40% compared to wild-type. Transgenic rods had slightly lower absolute sensitivity at 520 nm but higher relative sensitivity at long wavelengths compared to wild-type, which we attribute to the cone pigment. The significant spectral separation between the native rod (λmax ∼520 nm) and the transgenic cone (λmax ∼620 nm) pigments allowed us to estimate transgene expression levels by fitting the measured spectral sensitivity with a combination of A2 spectral templates. Expression varied among animals as well as within the same retina, and was on average 5% of the wild-type rhodopsin. Transgenic rods exhibited a high level of dark noise which varied linearly with cone pigment expression level. On average, the noise in transgenic rods was 16 times higher than in wild-type rods but still significantly lower than expected from previous noise measurements in salamander red cones. Conclusion: We have demonstrated for the first time the functional expression of a cone visual pigment in rods. The expressed cone pigment induces a red shift in the spectral sensitivity of rods and significantly increases their dark noise.
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