Acta2-GCaMP8.1-mVermilion transgenic mice express the GCaMP8.1/mVermilion fusion protein under control of the Acta2 locus promoter/enhancer regions within the BAC transgene. Acta2-GCaMP8.1-mVermilion mice from founder line B34-4 exhibit high expression levels in smooth muscle cells (e.g., blood vessels, lung airways, gut and bladder) - consistent with endogenous Acta2. Additional details described further below. Calcium is a key molecular signal for cell functions including heart, smooth muscle, vessel, and airway contraction, lung secretion, autonomic neurotransmission and immunocyte function. GCaMP8.1 is a sensitive GCaMP variant with high dynamic range and fast response kinetics, as well as improved calcium-binding affinity. The GCaMP8.1/mVermilion fusion protein has GCaMP8.1 fused in-frame at its C-terminus to the fluorescent protein mVermilion (an mCherry derivative with approximately 2-fold brighter emission). In the absence of calcium binding, low/baseline EGFP fluorescence and high mVermilion fluorescence are observed. Following calcium binding, bright EGFP fluorescence is observed at several-fold greater levels than baseline (see additional GCaMP8 information below) and the high mVermilion fluorescence continues. Because mVermilion fluorescence should be relatively constant (independent of calcium changes), GCaMP8.1/mVermilion is useful for ratiometric measurements of calcium signaling. For these Acta2-GCaMP8.1-mVermilion transgenic mice, the donating investigator reports that expression patterns appear to mimic the endogenous Acta2 expression (smooth muscle cells in blood vessels, lung airways, gut and bladder). Specifically, GCaMP8.1/mVermilion expression in smooth muscle cells in vessels of the brain, heart and spleen was demonstrated via direct fluorescence (both green and red). As expected, dim EGFP fluorescence and bright mVermilion fluorescence is observed in the absence of calcium binding, whereas bright EGFP fluorescence and high mVermilion fluorescence is observed after calcium binding. They have not tested expression in other tissues to date (July 2019). Mice hemizygous for the Acta2-GCaMP8.1-mVermilion transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. To date (July 2019), it has not been attempted to make this strain homozygous. The genetically encoded calcium indicator GCaMP8.1 is a codon-optimized variant of GCaMP8 having one additional mutation (M144I) in its calmodulin region for improved calcium-binding affinity (lowers the Kd from 235 nM to 188 nM). GCaMP8 is a sensitive GCaMP variant with high dynamic range and fast response kinetics (<a href="http://www.ncbi.nlm.nih.gov/pubmed/23240011">Ohkura et al. 2012 PLoS One 7:e51286</a>). Specifically, the high signal amplitude and low baseline fluorescence of GCaMP8 results in ability to detect single Ca2+ spikes / single neuronal action potentials. Compared to GCaMP3, the GCaMP8 has ~3-fold greater dynamic range, similar response kinetics, ~2-fold more rapid decay kinetics and lower baseline fluorescence. 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 bright EGFP fluorescence is observed at several-fold greater levels than baseline (e.g., ~16-fold greater in neurons ex vivo). In the presence of Ca2+, GCaMP8 has absorbance and emission peaks of ~488 nm and ~510 nm, respectively. This mouse model is available by way of a collaborative effort between Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (<a href="http://chromus.vet.cornell.edu">CHROMus</a>) and The Jackson Laboratory.

Acta2-GCaMP8.1-mVermilion transgenic mice express the GCaMP8.1/mVermilion fusion protein in smooth muscle cells (e.g., blood vessels, lung airways, gut and bladder). GCaMP8.1 is a sensitive GCaMP variant with high dynamic range and fast response kinetics, as well as improved calcium-binding affinity. Upon GCaMP8.1 binding to calcium, increased EGFP fluorescence is observed. Because mVermilion fluorescence should be relatively constant (independent of calcium changes), these Acta2-GCaMP8.1-mVermilion mice are useful for ratiometric measurements of calcium signaling in smooth muscle cells both in vivo and in vitro.

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