Ai94(TITL-GCaMP6s)-D;CaMK2a-tTA (or Ai94D;CaMK2a-tTA) mice have a Cre/Tet-dependent, fluorescent calcium indicator GCaMP6s (an ultrasensitive detector of single neuronal action potentials with slower decay and 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, Ai94D;CaMK2a-tTA mice are designed to have tTA expression in forebrain neurons but GCaMP6s expression (EGFP fluorescence) blocked by a floxed STOP cassette. Of note, the similarly designed Ai62(TITL-tdT)-D reporter (Stock No. 022731) combined with the same Camk2a-tTA transgene (Stock No. 007004) had offspring reported to have a few very weakly tdTomato-positive cells in cortex and hippocampus.
Following exposure of Ai94D;CaMK2a-tTA mice to Cre recombinase, the floxed STOP cassette in Ai94D is deleted; leading to expression of GCaMP6s in forebrain neurons. GCaMP6s expression can be regulated with tetracycline or its analog doxycycline (dox).

When Ai94D;CaMK2a-tTA mice are bred with Cre-driver lines to create triple transgenic animals (GCaMP6s+/tTA+/Cre+), EGFP fluorescence in cre-expressing cells is low in the absence of calcium binding. Following calcium binding (such as neuronal activation), increased EGFP fluorescence is observed in those cells. See further phenotype considerations below.

Ai94D;CaMK2a-tTA mice heterozygous or homozygous for Ai94D and hemizygous for CaMK2a-tTA are viable and fertile with no reported gross physical or behavioral abnormalities. To date (October 2016), it has not been attempted to make Ai94D;CaMK2a-tTA animals homozygous for the CaMK2a-tTA transgene.

For characterization information, see images at the Allen Institute for Brain Science website (Ai94(TITL-GCaMP6s) images).

Further phenotype considerations: Steinmetz et al. 2017 ENeuro reports aberrant neural activity is occasionally observed in Emx1-Cre;Camk2a-tTA;Ai94 mice. This paper is summarized below.
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 GCaMP6s: The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). Compared to GCaMP6f, the slower kinetics render GCaMP6s more sensitive and slower to decay. GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (with several amino acid substitutions designed to optimize dynamic range, baseline fluorescence and sensitivity), and a rat calmodulin sequence modified to increase the fluorescence change for small calcium transients. 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 and disrupting the genes Vipr2 (vasoactive intestinal peptide receptor 2), Wdr60 (WD repeat domain 60), Esyt2 (extended synaptotagmin-like protein 2), Ncapg2 (non-SMC condensin II complex, subunit G2), and Ptprn2 (protein tyrosine phosphatase, receptor type, N polypeptide 2). The deletion results in a functional knock-out of Vipr2, Wdr60, Esyt2, and Ncapg2 in homozygous mice. Founder line 1 has a copy number of greater than 20 [Goodwin et al. 2019 Genome Res. 29:494].