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J. Chen, M. Wu, S. A. Sezate, J. F. McGinnis; Light-Induced Cone Alpha-Transducin Translocation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):70.
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Transducin, a G-protein, is involved in the phototransduction cascade in photoreceptor cells. The alpha subunit of Transducin (Tα) is membrane associated and located in the outer segments of rods in the dark whereas it is soluble and located in the inner segment of rods in the light. Cone Tα is compartmentalized in the outer segment but does not translocate. We hypothesized that the unique amino acid sequence of each protein, or some cell-specific component, was responsible for the difference between the compartmentation and response to light of rTα and cTα. To test our hypothesis, we established a transient transgenic strategy to knock-in mRNAs using the in vivo transient expression of cDNA constructs in non-viral vectors. The cone Tα was exogenously expressed within rod photoreceptor cells and the location of the cone Tα within rods were examined under different light conditions.
The cDNA sequence of mouse cone Tα was cloned into pIRES2-EGFP vector which expresses cone Tα and green fluorescent protein bicistronically under control of the CMV promoter. Vector DNA in saline was injected subretinally into the eyes of neonatal rats followed by electroporation. Control pups were injected with the same amount of vehicle. Transfection efficiency was indicated by green fluorescence; and the location of cone Tα was determined by immunofluorescence microscopy.
Injection of the cDNA constructs resulted in the successful transient transfection of retinal cells. The green fluorescence expression was detected as early as day 14 up to the day 60, the last age tested. With the CMV promoter, approximately 95% of the transfected cells were photoreceptor cells while the remaining 5% were located within the inner nuclear layer and, rarely, within the ganglion cell layer. When cone Tα was exogenously expressed in rods, its localization paralleled that of endogenous rod Tα under light and dark conditions.
We successfully established an in vivo transient retinal transfection model with non-viral vector constructs. The data demonstrate that proteins of interest can be knocked in and that their functional involvement in translocation can be evaluated. The data also suggest that the rods contain cellular components sufficient to translocate and compartmentalize cone Tα in response to light.
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