The Snap25-2A-GCaMP6s-D knockin allele has a viral 2A oligopeptide sequence (T2A) that mediates ribosomal skipping and a GCaMP6 slow variant calcium indicator (GCaMP6s), all inserted downstream of the synaptosomal-associated protein 25 (Snap25) coding region. This is designed to have widespread brain expression of the GCaMP6 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics) that results in EGFP expression following calcium binding (such as neuronal activation). 

Functional testing/characterization of Snap25-2A-GCaMP6s-D mice has not yet been completed (October 2014). While they are expected to have pan-neuronal GCaMP6s expression, native fluorescence and immunohistochemical staining (via anti-GFP antibody) are not yet defined. Like other GCaMP6s-expressing mice from the same donating investigator, Snap25-2A-GCaMP6s-D cells can be expected to have low EGFP expression throughout the brain in the absence of calcium binding. Following calcium binding (such as neuronal activation), significantly increased EGFP expression is expected. Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. To date (August 2017), it has not been attempted to generate homozygous mice by the donating investigator or The Jackson Laboratory.

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.

 Snap25-2A-GCaMP6s-D knockin mice are a calcium-indicator tool strain. These mice are designed to have widespread brain expression of the GCaMP6 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics) that results in EGFP expression following calcium binding (such as neuronal activation). These mice may also be useful for studying t-SNARE proteins and synaptic vesicle/plasma membrane fusion.

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