Ai93(TITL-GCaMP6f)-D;CaMK2a-tTA (or Ai93D;CaMK2a-tTA) mice have a Cre/Tet-dependent, fluorescent calcium indicator (GCaMP6f; a detector of single neuronal action potentials with fast response kinetics) inserted into the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated), as well as a transgene directing tetracycline-controlled transactivator protein (tTA) expression in forebrain neurons.

In the absence of Cre recombinase, Ai93D;CaMK2a-tTA mice are designed to have tTA expression in forebrain neurons but GCaMP6f expression (EGFP fluorescence) is blocked by a floxed STOP cassette. Following exposure to Cre recombinase, the floxed STOP cassette in Ai93D is deleted; leading to expression of GCaMP6f in forebrain neurons. GCaMP6f expression can be regulated with tetracycline or its analog doxycycline (dox).

When Ai93D;CaMK2a-tTA mice are bred with Cre-driver lines to create triple transgenic animals (GCaMP6f+/tTA+/Cre+), EGFP fluorescence in cre-expressing cells is low in the absence of calcium binding. Following calcium binding (such as neuronal activation), bright EGFP fluorescence is observed in cells expressing Cre recombinase. See further phenotype considerations below.

The donating investigator also reports that following neuronal activation, the fluorescent intensity observed in triple transgenic Ai93D mice (GCaMP6f+/tTA+/Cre+) is significantly greater than that of the double transgenic Ai95D system (GCaMP6f+/Cre+ ; see Stock No. 024105). Although both systems utilize the same GCaMP6f gene, they differ in chromosomal location and Tet-inducibility.

Ai93D;CaMK2a-tTA mice heterozygous for both targeted mutations are viable and fertile with no reported gross physical or behavioral abnormalities. The phenotype of mice homozygous for both Ai93D and CaMK2a-tTA has not been characterized to date (April 2014).

Further phenotype considerations: Steinmetz et al. 2017 ENeuro reports aberrant neural activity resembling interictal spikes were observed in some genotypes of GCaMP6f-expressing Ai93 mice. This activity was most prominent in Emx1-Cre;Camk2a-tTA;Ai93 mice and Emx1-Cre;Rosa26-ZtTA;Ai93 mice, and also in Camk2a-tTA;Ai93 mice that had undergone germline Cre recombination. Aberrant neural activity was also occasionally observed in Slc17a7-IRES-Cre;Camk2a-tTA;Ai93 mice and GCaMP6s-expressing Emx1-Cre;Camk2a-tTA;Ai94 mice. Several other combinations were absent of aberrant neural activity - including Emx1-CreERT2;Camk2a-tTA;Ai93 maintained on doxycycline, Camk2a-tTA;Ai93 with other Cre-drivers (Rorb-IRES2-Cre or Scnn1a-Tg3-Cre), Camk2-tTA with another GCaMP6s transgene (Camk2a-tTA;TRE-GCaMP6s line G6s2), Emx1-driven GCaMP6f (Emx1-Cre;Ai95) or GCaMP6s (Emx1-Cre;Ai96), excitatory neuron-expressing GCaMP6s strains (Snap25-2A-GCaMP6s and Thy1-GCaMP6s line GP4.3) and Pvalb-driven channelrhodopsin2 (Pvalb-Cre;Ai32).

Note on genetic background effect on tTA-induced neurotoxicity: Han et al. 2012 J Neurosci. 32:10574 reports mice expressing Tg(CaMK2a-tTA)1Mmay on the C57BL/6 background exhibit resistance to tTA-induced neurotoxicity (dentate gyrus granule cell layer atrophy). All other backgrounds tested (FVB/NJ, CBA/J, 129X1/SvJ, C3H/HeJ and DBA/1J) exhibit varying levels of neurotoxicity.

Note on Ai93D allele design: Prior to recombination, Ai93D mice have the TIGRE-Ins-TRE-LSL-GCaMP6f conditional allele, designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette (LSL) upstream of GCaMP6f. Ai93D genotyping/sequencing revealed that two tandem copies of the TRE::floxed-STOP::GCaMP6f replacement vector were inserted during gene targeting. Therefore, the Ai93D allele is structurally composed of TIGRE-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttB-PGK-5'hygro-SD-frt5-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttL. The donating investigator reports that crossing Ai93D mice (Stock No. 024103) to several different Cre mouse lines consistently produces offspring with the expected GCaMP6f expression. Sufficiently strong and prolonged expression of Cre (as in the case of Cre-expressing mice such as CaMK2a-tTA) is likely to recombine between the furthest loxP sites; resulting in one copy of Tet-inducible TRE::GCaMP6f. While the floxed-STOP regions may individually recombine with weaker and/or transient Cre expression, the donating investigator suggests the Tet-dependent calcium activity monitoring function should not be adversely affected in those recombination scenarios (i.e., two copies of Tet-inducible TRE::GCaMP6f, or one copy of Tet-inducible TRE::GCaMP6f with the other copy retaining the floxed-STOP region).

Note on GCaMP6f: The GCaMP6 fast variant calcium indicator (GCaMP6f) is a detector of single neuronal action potentials with fast response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. Compared to GCaMP6s, GCaMP6f has faster response but is less sensitive and faster to decay. The GCaMP6f fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). GCaMP6f has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (with several amino acid substitutions designed to increase dynamic range and baseline fluorescence), and a rat calmodulin sequence modified to increase the fluorescence change for small calcium transients and accelerated kinetics. Other modifications in the cpEGFP-to-CaM linker also improve sensor function. 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.

As described for Stock No. 007004, the CaMK2a-tTA transgene integrated into chromosome 12 causing a 508.12 Kb deletion that spans the 3' half of Vipr2 (vasoactive intestinal peptide receptor 2), the entire Wdr60 (WD repeat domain 60), Esyt2 (extended synaptotagmin-like protein 2), D430020J02Rik (RIKEN cDNA D430020J02 gene) and Ncapg2 (non-SMC condensin II complex, subunit G2) loci and the first two exons of Ptprn2 (protein tyrosine phosphatase, receptor type, N polypeptide 2). Homozygous mice will therefore have a functional knock-out of the deleted loci (Wdr60, Esyt2, D430020J02Rik, Ncapg2), and altered or null expression of Vipr2 and Ptprn2. Founder line 1 has a copy number of greater than 20 [Goodwin et al. 2019 Genome Res. 29:494].