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



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Public Library of Science



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.



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


NIH grants (NS083702, NS094224 and NS092297).


CC BY 4.0 (Attribution), ©2019 The Authors