The R26-LSL-SOUL-P2A-tdT knock-in allele has a CAG hybrid promoter, loxP-flanked STOP cassette and bicistronic SOUL-P2A-tdTomato sequence. Prior to Cre recombinase exposure, this allele is designed to have the floxed-STOP cassette preventing expression of the two individual downstream genes - a step-function opsin with ultra-high light sensitivity (SOUL; see detailed description below) and a tdTomato fluorescent protein. Following exposure to Cre recombinase, the floxed-STOP cassette is deleted - resulting in SOUL expression and tdTomato fluorescence. Subsequent illumination of SOUL-expressing cells with blue light (~470 nm) leads to stable step in membrane potential (depolarization) in these cells - and the channel can remain in the conducting state for more than 30 minutes, during which time it can be deactivated using orange light (~590 nm). [<a href="https://pubmed.ncbi.nlm.nih.gov/32353253">Gong et al. 2020 Neuron 107:38 (PMID:32353253)</a>] Specifically, the donating investigator reports in the absence of Cre, mice heterozygous for R26-LSL-SOUL-P2A-tdT exhibit no fluorescence, no abnormal action potentials/depolarizations, and are viable and fertile with no reported gross physical or behavioral abnormalities. Homozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. Further characterization of homozygotes has not been performed to date (May 2020). When R26-LSL-SOUL-P2A-tdT mice are bred with C57BL/6J-congenic PV-Cre (Stock No. <a href="https://www.jax.org/strain/017320">017320</a>), the donating investigator observed robust/bright tdTomato fluorescence in the resulting PV-Cre:SOUL animals that was consistent with the expected Cre expression pattern. Before blue light, they observe no action potential among the cells recorded. Blue light activation of SOUL occurs at levels sufficient to effectively induce stable step in membrane potential, but does not directly induce action potential firing activity. Orange light illumination of SOUL-expressing neurons leads to rapid deactivation of the channel, in contrast to the very slow spontaneous channel deactivation time. The tdTomato expression remains the same as before illumination. When bred with C57BL/6J-congenic ChAT-Cre mice (originally from Stock No. <a href="https://www.jax.org/strain/006410">006410</a>), the donating investigator reports tdTomato fluorescence was observed in the resulting ChAT-Cre:SOUL animals that was consistent with the expected Cre expression pattern (although fluorescence was dimmer compared to PV-Cre:SOUL mice). SOUL expression levels may be confirmed by 2A antibody staining. While the cytoplasmic tdTomato fluorescence facilitated visualization of SOUL-expressing neurons, the P2A-mediated self-cleavage physically uncoupled tdTomato from SOUL (as identified by the 2A fragment remaining at the SOUL C-terminus) and thus prevented the possible interference of the fluorophore with the opsin's function. The expression/function of SOUL and tdTomato has been tested in cortical neuronal cultures, acute slices and in vivo, although tissues other than brain have not yet been evaluated by the donating investigator (August 2020). Of note, frt sites in the R26-LSL-SOUL-P2A-tdT knock-in allele allow for additional targeted replacement of the LSL-SOUL-P2A-tdT sequences through FLP-mediated recombination if so desired. Similarly, the AttB/AttP-flanked PGK-frt-Neo-polyA cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired. 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. Chlamydomonas reinhardtii-derived channelrhodopsin-2 (ChR2 or COP4) is a blue light-driven cation channel that depolarizes the cell and causes action potentials. The step-function opsin with ultra-high light sensitivity (SOUL; COP4*C128S*D156A*T159C) is a ChR2 variant modified to have three amino acid substitutions that result in step function-like properties with extended channel open state and enhanced photocurrent amplitudes. As such, illuminating SOUL-expressing cells with blue light (~470 nm) leads to stable step in membrane potential (depolarization) in these cells - and the channel can remain in the conducting state for more than 30 minutes, during which time it can be deactivated using orange light (~590 nm). Because of its long deactivation kinetics, SOUL may be particularly useful for manipulating cellular activity in conditions when prolonged connection to a fiber optic cable is not desired. The extreme light sensitivity of SOUL may also be advantageous in some applications (e.g., non-invasive stimulation from outside the skull), but may be a disadvantage in others (e.g., projection-specific modulation of neural activity).

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R26-LSL-SOUL-P2A-tdT mice are a Cre-dependent, optogenetic line with a loxP-flanked STOP cassette preventing transcription of a step-function opsin with ultra-high light sensitivity (SOUL; a channelrhodopsin modified to have step function-like properties with extended channel open state and enhanced photocurrent amplitudes) and a tdTomato fluorescent protein. After Cre recombinase exposure, subsequent illumination of SOUL-expressing cells with blue light (~470 nm) leads to stable step in membrane potential (depolarization) in these cells - and the channel can remain in the conducting state for more than 30 minutes, during which time it can be deactivated using orange light (~590 nm).

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B6;129-Gt(ROSA)26Sor<tm2(CAG-COP4*C128S*D156A*T159C,-tdTomat