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Hiroshi Tomita, Eriko Sugano, Hitomi Isago, Namie Murayama, Zhuo Wang, Makoto Tamai; Improvement Of Wavelength Sensitivities By The Modification Of Volvox Channelrhodopsin-1 Gene. Invest. Ophthalmol. Vis. Sci. 2011;52(14):478.
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It has been reported that the intravitreal injection of the channelrhodopsin-2 (ChR2) gene into genetically blind rats restored visual response. However the sensitive wavelength of ChR2 is limited below 540 nm. In this study, we modified a volvox derived channelrhodopsin-1 (VChR1) gene based on a bioinfomatic approach to improve wavelength sensitivities and expression profiles in mammalian cells.
We made various types of modified VChR1 genes by the replacement of the N-terminal or C-terminal fragment of VChR1 gene with other proteins. The modified VChR1 genes were electroporated into cultured HEK293 cells to investigate the expression profiles. The photocurrents were recorded under the whole-cell patch clamp of a conventional system. We also made an adeno-associated virus vector type 2 including the modified VChR1 gene fused to a fluorescent protein, Venus (AAV-mVChR1V) and intravitreously injected into Royal College of Surgeons (RCS) rats. Visually evoked potentials (VEPs) were recorded 2 months later.
The mVChR1 which is chimeric protein with the chlamydomonas derived-channelrhodopsin-1 showed the diffused expression in the cell body and plasma membrane. The photocurrents were evoked by 1-s pulse illumination of wavelength between 400 and 600 nm. The VEPs in mVChR1-transferred RCS rats were also recorded by the stimulus wavelength from 450nm to 600nm. The mVChR1 was more sensitive to the white flash than the chlamydomonas derived channelrhodopsin-2. The expression of mVChR1 in the retina were mainly observed in retinal ganglion cells, which was almost same as those of ChR2.
The mVChR1 showed wide range spectrum sensitivities. The gene transfer of ChR2 with mVChR1 into blind rats will increase the visible wavelength.
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