Leaky Expression of Channelrhodopsin-2 (ChR2) In Ai32 Mouse Lines

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Public Library of Science

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Abstract

Optogenetics enables the selective activation of genetically-targeted neuronal populations using light-sensitive ion channels. Genetic strategies using Cre-dependent mouse strains, especially the Ai32 line expressing Channelrhodopsin (ChR2)-EYFP fusion protein, have been a popular means to drive opsin expression in a cell-type specific manner. Here we report a low level of leaky ‘off-target’ (Cre-independent) ChR2-EYFP expression in Ai32/ Ai32 homozygous mice throughout the nervous system. This leaky off-target expression was characterized in multiple prevalent nervous system regions using anti-EYFP immunostaining. Expression of full-length ChR2-EYFP protein was confirmed using immunoprecipitation followed by Western blotting. Notably, light stimulation of these ChR2-EYFP expressing neurons in the spinal cord dorsal horn did not induce detectable photocurrents in juvenile 4-week old mice. Given the wide use of the Ai32 line by many labs, our results suggest researchers should be vigilant of possible off-target ChR2-EYFP expression in their region of interest, especially when generating Ai32/Ai32 homozygotes to drive high levels of ChR2-EYFP expression in adult mice.

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Channelrhodopsin, Channelrhodopsin 2, Adulthood, Age groups, Animal tissues, Homozygote, Immunohistochemistry, Immunoprecipitation, Mice, Nervous system, Spinal Cord Dorsal Horn, Western immunoblotting

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NIH grants (NS083702, NS094224 and NS092297).

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CC BY 4.0 (Attribution), ©2019 The Authors

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