Ai32 mice homozygous for the Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream ChR2(H134R)-EYFP fusion gene. Because this CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, ChR2(H134R)-EYFP expression is determined by which tissue(s) express Cre recombinase. When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissues; resulting in expression of the ChR2(H134R)-EYFP fusion protein. ChR2(H134R)-EYFP expression following exposure to cre can be detected by EYFP fluorescence (and presumably by mRNA [in situ hybridization] and antibody staining [immunohistochemistry]; although this was not tested by the donating investigator). The donating investigator reports Ai32 mice have no significant expression of ChR2(H134R)-EYFP prior to introduction of Cre recombinase. Importantly, very low levels of ChR2(H134R)-EYFP expression may be present before Cre recombination - but the ChR2(H134R)-EYFP expression levels after Cre recombination are significantly greater than those baseline levels. As such, it is recommended that researchers include Cre-negative Ai32 controls to establish the baseline ChR2(H134R)-EYFP levels in their experiments. For characterization information, see images at the Allen Institute for Brain Science website (<a href="http://connectivity.brain-map.org/transgenic/imageseries/list/1.html?gene_term=Ai32">Ai32 images</a>). Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired. Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired. The ChR2(H134R)-EYFP fusion protein is composed of a Chlamydomonas reinhardtii-derived channelrhodopsin-2 that harbors a gain-of-function H134R substitution fused in-frame with an enhanced yellow fluorescent protein. The ChR2(H134R) is designed to cause larger stationary photocurrents compared to ChR2. 
 The bacterial opsins are retinal-binding proteins that combine a light-sensitive domain with an ion channel or pump; providing light-dependent ion transport, membrane potential alteration, and sensory functions to bacteria. This ChR2(H134R) functions as a blue light-driven cation channel that depolarizes the cell and causes action potentials. As such, illuminating ChR2(H134R)-expressing cells with blue light (450-490 nm) leads to rapid and reversible photostimulation of action potential firing activity in these cells.

Ai32 mice express an improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase. These mice can be used in optogenetic studies for rapid in vivo activation of excitable cells by illumination with blue light (450-490 nm).

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