Leaky expression of channelrhodopsin-2 (ChR2) in Ai32 mouse lines

PLoS One. 2019 Mar 26;14(3):e0213326. doi: 10.1371/journal.pone.0213326. eCollection 2019.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Channelrhodopsins / genetics
  • Channelrhodopsins / metabolism*
  • Homozygote
  • Light
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Mice
  • Neurons
  • Optogenetics*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*

Substances

  • Bacterial Proteins
  • Channelrhodopsins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria