Ai96(RCL-GCaMP6s) mice (also called Ai96) harbor the Rosa-CAG-LSL-GCaMP6s conditional allele, designed with a floxed-STOP cassette upstream of the GCaMP6 slow variant calcium indicator (GCaMP6s; see detailed description below). Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of GCaMP6s is prevented by STOP cassettes. After exposure to Cre recombinase, EGFP fluorescence is observed following calcium binding (such as neuronal activation). 
 Specifically, the donating investigator reports Ai96 mice have no reported levels of EGFP fluorescence prior to exposure to Cre recombinase. When Ai96 mice are bred with a Cre-driver line to create double transgenic animals (GCaMP6s+/Cre+), cells expressing Cre exhibit low EGFP fluorescence in the absence of calcium binding. Following calcium binding (such as neuronal activation), increased EGFP fluorescence is observed in these cells. 
 The donating investigator also reports that following neuronal activation, the fluorescent intensity observed in double transgenic Ai96 mice (GCaMP6s+/Cre+) is less than that of the double transgenic Ai95 system using GCaMP6 fast variant calcium indicator (GCaMP6f+/Cre+ ; see Stock No. <a href="https://www.jax.org/strain/024105">024105</a>). Although both systems utilize Cre-dependent GCaMP6/EGFP fusion genes, they differ in the kinetics and sensitivity of their GCaMP6 variants. 
 Heterozygous Ai96 mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous Ai96 mice to date (March 2014). 
 For characterization information, see images at the Allen Institute for Brain Science website (<a href="http://connectivity.brain-map.org/transgenic/imageseries/list/1.html?gene_term=Ai96">Ai96(RCL-GCaMP6s) images</a>). 
 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/?term=23868258">Chen et al. 2013 Nature 499:295</a>), 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.

This Ai96 allele is available on a C57BL/6J genetic background as Stock No. <a href="https://www.jax.org/strain/028866">028866</a> and that version will replace this mixed background strain in the future. Ai96(RCL-GCaMP6s) (also called Ai96) mice are a Cre-dependent, fluorescent, calcium-indicator tool strain. Ai96 has a floxed-STOP cassette preventing transcription of the GCaMP6 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics). After Cre exposure, increased EGFP fluorescence is observed following calcium binding (such as neuronal activation).

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B6;129S6-Gt(ROSA)26Sor<tm96(CAG-GCaMP6s)Hze>/J