Ai163(TIT2L_GCaMP6s_ICL_tdTomato_P2A_tTA2_WPRE)-D (also called Ai163D) mice harbor the pTRE2-LSL-GCaMP6s-CAG-LSL-tdTom-tTA2 conditional allele, designed with a TRE2 promoter, loxP-flanked STOP cassette and GCaMP6s sequence, and then a CAG promoter, lox2272-flanked STOP cassette, tdTomato, P2A, and tTA2 sequence. Prior to Cre recombinase exposure, this allele is designed to have the two floxed-STOP cassettes preventing expression of the downstream genes (GCaMP6s, tdTomato, and tTA2). When bred to mice expressing Cre recombinase, the offspring will have both floxed-STOP cassettes deleted in the cre-expressing cells/tissues - resulting in GCaMP6s, tdTomato, and tTA2 expression. When GCaMP6s is expressed, subsequent calcium binding (such as neuronal activation) results in robust EGFP fluorescence. Further administration of tetracycline (or its analog doxycycline [dox]) will directly inhibit tTA2 activity, and therefore is expected to reduce GCaMP6s expression levels. Specifically, the donating investigator reports Ai163D exhibit no native fluorescence in the absence of Cre. When Ai163D mice are bred with neocortical layer-specific Cre-driver lines (e.g., Calb1-IRES2-Cre [see Stock No. <a href="https://www.jax.org/strain/028532">028532</a>]) or interneuron Cre-driver lines (e.g., Sst-IRES2-Cre [see Stock No. <a href="https://www.jax.org/strain/013044">013044</a>]) result in double transgenic mice with robust native GCaMP6s expression. Ai163D 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. The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics (<a href="http://www.ncbi.nlm.nih.gov/pubmed/23868258">Chen et al. 2013 Nature 499:295</a>), and is an improved version of GCaMP5G. Compared to GCaMP6f, the slower kinetics render GCaMP6s more sensitive and slower to decay. In the absence of calcium binding, dim EGFP fluorescence is expected as there is a pore from the outside of its barrel into the chromophore. Upon calcium binding, this pore becomes occupied and fluorescence is increased. The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with M153K, V163A, S175G, D180Y, T203V, A206K and V93I mutations to increase dynamic range/baseline fluorescence, as well as K166H mutation for increased sensitivity/slower kinetics]), and a rat calmodulin DNA fragment (CaM; aa 2-148 with N60D, D78Y, T79R, S81T and R90G mutations [also called N362D, D380Y, T381R, S383T and R392G, respectively] to increase the fluorescence change for small calcium transients. The T302L and R303P amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function.

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Ai163(TIT2L_GCaMP6s_ICL_tdTomato_P2A_tTA2_WPRE)-D (also called Ai163D) mice are a Cre-dependent, Tet-controllable, fluorescent calcium-indicator tool strain - created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Exposure to Cre recombinase removes both STOP cassettes, resulting in expression of tTA2, tdTomato, and GCaMP6s (an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics). GCaMP6s expression may then be diminished by doxycycline.

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