Prabhakar, ArthiVujovic, D.Cui, L.Olson, W.Luo, W.2019-12-182019-12-182019-03-261932-6203https://hdl.handle.net/10735.1/7132Optogenetics 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.enCC BY 4.0 (Attribution)©2019 The Authorshttp://creativecommons.org/licenses/by/4.0/ChannelrhodopsinChannelrhodopsin 2AdulthoodAge groupsAnimal tissuesHomozygoteImmunohistochemistryImmunoprecipitationMiceNervous systemSpinal Cord Dorsal HornWestern immunoblottingarticlePrabhakar, A., D. Vujovic, L. Cui, W. Olson, et al. 2019. "Leaky expression of channelrhodopsin-2 (ChR2) in Ai32 mouse lines." PLOS One 14(3): art. e0213326, doi: 10.1371/journal.pone.0213326143