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H. Tomita, E. Sugano, H. Isago, H. Yawo, T. Ishizuka, M. Tamai; Improvement of Light Sensitivity in the ChR2-Expressing Rat Retinas By Co-Expression of Fluorescent Protein. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5202. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To increase the light sensitivity in ChR2 transferred rat retinas, a fluorescent protein which has the emission peak at a sensitive wavelength of the ChR2 was co-expressed.
The N-terminal fragment of the ChR2 protein was fused to various fluorescent protein which have different emission peaks; Cyan fruorescent protein (CFP), Venus or mCheryy. They were introduced into the plasmid vector driven by the CAG promoter to produce the adeno-associated virus (AAV) vectors. Each AAV vector (102-103 particle /ml) was intravitreouslly injected into 6 month old Royal Colleage of Surgeons (RCS) rat retinas. Efficiencies of the gene transduction were evaluated by counting the fluorescent cells in flat-mounted retinas 12 weeks after the injection. Visually evoked potentials (VEPs) were recorded 8 weeks after the injection.
There was no difference of the transduction efficiencies among the fused fluorescent protein (CFP, Venus, and Cherry). The gene expression was observed in about 30 % of total ganglion cells, and in some of the inner nuclear layer. Though 6-month old RCS rats have no response to the light stimulation at any intensities using blue LED before the injection, VEPs were recorded in all rats transferred the ChR2 fused fluorescent protein by the light intensities at more than 500 lux. The rats transferred the ChR2-CFP have the response to blue LED stimulation at less than 500 lux.
Co-expression of CFP could increase the light sensitivity of ChR2. ChR2 may receive photon not only directly from blue LEDs but also from the emission of the fused CFP.
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