Ai167D mice harbor the TIGRE-Ins-tetO-LSL-ChrR-tdT-Ins-CAG-LSL-tTA2 conditional allele, designed with a tetO promoter, loxP-STOP-loxP cassette and ChrR-tdT sequence, and then a CAG promoter, lox2272-STOP-lox2272 cassette and tTA2 sequence. ChrR-tdT is a light-gated cation channel that can be used as an optogenetic tool to activate neurons fused to the tdTomato fluorophore - see additional information below. Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (ChrR-tdT and tTA2). Ai167D heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (August 2024), it has not been attempted to make this strain homozygous. When bred to mice expressing Cre recombinase, the offspring will have both STOP cassettes deleted in the cells where cre is expressed - resulting in ChrR-tdT expression and tTA2 expression. Specifically, the donating investigator reports Ai167D exhibits no visible ChrR-tdT fluorescence in the absence of Cre. The donating investigator reports when Ai167D mice are bred with neocortical layer-specific and Cre-driver lines to create double mutant animals, robust native neocortical layer ChrR-tdT expression is evident as is expression across cortical layers II-V. However, the expression and survival is dependent of the driver lines used as there are caveats for use with certain driver lines. (see below). Combining Ai167D with Gad2-IRES-Cre mice (Stock No. <a href="https://www.jax.org/strain/010802">010802</a>) or Slc17a6-IRES-Cre mice (see Stock No. <a href="https://www.jax.org/strain/016963">016963</a>) results in embryonic lethality. When bred to Penk-IRES2-Cre-neo mice (Stock No. <a href="https://www.jax.org/strain/025112">025112</a>), very few double positive offspring are produced and those that survive have high morbidity and do not thrive. When bred with Sst-IRES-Cre mice (Stock No. <a href="https://www.jax.org/strain/013044">013044</a>), older animals develop ulcerative dermatitis. High morbidity and mortality above p80 are also observed. When crossed to Drd1-Cre mice or Adora2a-Cre mice, offspring exhibit abnormal phenotypes including small size, hydrocephalus, microphthalmia, abnormal legs, and small ears those that survive have high morbidity and do not thrive. Ai167D mice were successfully crossed with Cux2-CreERT2 mice, with cre/ERT2 expression throughout the central nervous system, including most brain regions and spinal cord, and the donating investigator saw expected expression after tamoxifen induction. ChrR-tdT is the result of a ChrimsonR coupled to the fluorescent reporter tdTomato. ChrimsonR (ChrR) is a light-gated cation channel that can be used as an optogenetic tool to activate neurons. It is optimally excited by light of approximately 590nm wavelength (aka, red-shifted). ChrimsonR is an artificial channelrhodopsin derived by introducing the K176R mutation into the alga-derived channelrhodopsin Chrimson. The mutation results in faster off-kinetics without altering the action spectrum.

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Ai167D (also called Ai 167(TIT2L_ChrimsonR_tdTomato_ICL_tTA2_WPRE)) is a cre recombinase responsive, Tet-controllable, fluorescent and optogenetic tool allele created by targeted insertion at the Igs7 locus (TIGRE). Exposure to Cre recombinase results in expression of tTA2 and ChrR-tdT (a light-gated cation channel; neuronal activation) in those cells where cre is expressed. ChrR-tdT expression may be expected to be diminished/eliminated by doxycycline.

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