Ai80(RCFL-CatCh)-D mice (also Ai80D) harbor the Rosa-CAG-FrtSTOPFrt-LoxSTOPLox-CatCh/EYFP conditional allele designed with both an frt-flanked STOP cassette and a loxP-flanked STOP cassette preventing transcription of the CatCh/EYFP fusion gene (ChR2(L132C)/EYFP; the calcium translocating, L132C mutant channelrhodopsin variant (CatCh) fused in-frame with an enhanced yellow fluorescent protein). Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of CatCh/EYFP is prevented by both STOP cassettes. After removal of both flanked STOP cassettes via Flp- and cre-mediated recombination, expression of CatCh/EYFP is expected in cells/tissues where the expression patterns of the individual promoters driving FLP recombinase and Cre recombinase overlap.
 Specifically, the donating investigator reports that Ai80D mice have no CatCh/EYFP expression (EGFP fluorescence) prior to introduction of both FLP and Cre recombinase. Weak, membrane-localized EYFP fluorescence is observed after deletion of both STOP cassettes. The donating investigator also reports that order in which the flanked STOP cassettes are removal is arbitrary. Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities.
 These Ai80D mice have not been fully characterized to date (September 2014). Whether CatCh/EYFP expression levels are measurable by mRNA (in situ hybridization) or antibody staining (immunohistochemistry) is not yet known. Functional testing has not yet been performed to test if light-induced opsin activation occurs at levels sufficient to effectively depolarize/activate cortical neurons. The donating investigator has not attempted to generate homozygous mice.
 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 calcium translocating channelrhodopsin (CatCh) is a ChR2 variant modified to have an L132C amino acid substitution for enhanced Ca2+ permeability; resulting in accelerated response time and a voltage response that is ~70-fold more light sensitive than wildtype ChR2. As such, illuminating CatCh-expressing cells with blue-to-green light (~450-530 nm; max 474 nm) leads to rapid and reversible photostimulation of robust action potential firing activity in these cells. Because CatCh activation is possible at naturally occurring light intensities while maintaining high temporal precision, it may also have applications in gene-therapeutic visual restoration efforts.

The Ai80(RCFL-CatCh)-D (or Ai80D) conditional allele has both an frt-flanked STOP cassette and a loxP-flanked STOP cassette preventing transcription of the calcium translocating channelrhodopsin (CatCh; an L132C mutant channelrhodopsin with enhanced Ca2+ permeability, accelerated response kinetics and ~70-fold greater light sensitivity) and EYFP. After removal of the flanked STOP cassettes by FLP- and Cre-recombination, CatCh/EYFP expression is expected in cells/tissues expressing both FLP recombinase and Cre recombinase. Subsequent illumination of CatCh-expressing cells with blue-to-green light leads to reversible photostimulation of action potential firing/neural activity in these cells.

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B6.Cg-Gt(ROSA)26Sor<tm80.1(CAG-COP4*L132C/EYFP)Hze>/J Frozen