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Hiroshi Tomita, Eriko Sugano, Kitako Tabata, Yoshito Watanabe, Taku Ozaki, Makoto Tamai; Ion Channel Properties of Cells Expressing Two Different Types of Channelrhodopsin Genes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5878.
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© ARVO (1962-2015); The Authors (2016-present)
Optogenetic technologies are expected to be applicable for clinical use in restoring vision. However, the degree of recovered visual function is highly dependent on the function of the chosen optogenetic gene. To investigate the effect on ion channel properties of cells expressing two different types of channelrhodopsin genes, we established a cell line expressing both a modified Volvox-derived channelrhodopsin gene (mVChR1) and channelrhodopsin-2 (ChR2).
An expression plasmid including ChR2 gene (pChR2-IRES-puro) was electroporated into cultured HEK293 cells. Following the establishment of HEK-ChR2 cell line, the linearized pAAV-mVChR1V vector was electroporated into stable transformant HEK-ChR2 cells. Venus-positive cells were sorted using a cell sorter as cells stably expressing the ChR2 and mVChR1 genes (HEK-ChR2+mVChR1). Photocurrents of these cell lines were recorded under whole-cell patch clamping of isolated cells. To investigate the effect on visual function of dual expression of genes with different wavelength sensitivities, we transduced a mVChR1 gene via an adeno-associated virus vector into transgenic rats harbouring the ChR2 gene in retinal ganglion cells. The native photoreceptor cells were degenerated by an intraperitoneal injection of N-methyl-N-nitrosourea prior to transduction of mVChR1.
The results of patch clamp recordings indicated that increased photocurrent mediated by dual gene expression was observed in wavelength ranges such as 550 and 600 nm, in which ChR2 did not exhibit effective functioning. In the ChR2 transgenic rats, visually evoked potentials were clearly detectable in spite of native photoreceptor function abolishment; however the responses were limited to within blue wavelengths. In contrast, the limited wavelength sensitivities were improved by the additional transduction of mVChR1, which exhibited sensitivities to green and red.
The transductions of dual genes encoding channelrhodopsins that exhibit different wavelength sensitivities represents a promising candidate method to expand and to enhance rescued wavelength sensitivities in blind subjects.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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