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.
Michael I Kotlikoff, Cornell University
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 (Ohkura et al. 2012 PLoS One 7:e51286). 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 (CHROMus) and The Jackson Laboratory.
Acta2-GCaMP8.1-mVermilion transgenic mice (also called Acta2-GCaMP8.1/mVermilion or Acta2BAC-GCaMP8.1-mVermilion) were designed in the laboratory of Dr. Michael I. Kotlikoff (Cornell University) as part of Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).
The ~175 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-370F21 was obtained; containing the entire actin alpha 2 locus (Acta2) as well as ~92 kbp of 5' flanking sequences (including the complete Fas locus) and ~68 kbp of 3' flanking sequences (including the complete Stambpl1 locus). Using homologous recombination/BAC recombineering, a 2993 bp GCaMP8.1/mVermilion-SV40pA construct (see details below) was inserted into the ATG start site of the BAC Acta2 gene (replacing the initiation codon of Acta2 in exon 2). No other loci on the BAC were altered. The 11,612 bp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette.
The resulting ~189.6 kbp modified BAC (Acta2BAC-GCaMP8.1-mVermilion) was purified and then microinjected into the male pronucleus of C57BL/6J x SJL/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Acta2-GCaMP8.1-mVermilion founder line B34-4 was identified with high sensor expression in smooth muscle cells. The donating investigator reports that Acta2-GCaMP8.1-mVermilion transgenic mice from this founder line were backcrossed to C57BL/6J for a total of at least six generations, and then hemizygous males (black coat color) were sent to The Jackson Laboratory Repository in 2019. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin). The transgene insertion site(s) and transgene copy number were not characterized (July 2019).
The GCaMP8.1-mVermilion fusion protein is composed of the GCaMP8.1 sequence fused at its C-terminal to the N-terminal of the mVermilion sequence via a flexible alanine-proline repeat linker (GSSTSG-APAPAPAPAPAPAP-SEF).
The genetically encoded calcium indicator GCaMP8.1 is a variant of GCaMP8 having one additional mutation (M144I) in its calmodulin region for improved calcium-binding affinity. GCaMP8 is a sensitive GCaMP variant with high dynamic range and fast response kinetics (Ohkura et al. 2012 PLoS One 7:e51286). First, Dr. Kotlikoff had the codon-optimized GCaMP8 sequence synthesized to have (from N-term to C-term) a polyHis plasmid leader sequence essential for thermal stability (RSET with the ΔH and ΔR2 mutations), a 20 residue peptide of chicken smooth muscle myosin light chain kinase (M13; target peptide for a Ca2+-bound CaM), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with mutations listed below]), a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations listed below]). The cpEGFP mutations result in improved brightness (D180Y and V93I), thermal stability (V163A and S175G), dimerization prevention (A206K), dynamic range/baseline fluorescence (M153K, T203V, N105Y and E124V), and overall functionality (S205N and I47F). The CaM mutations are for higher calcium affinity (M36L and N60D) and greater signal increase (N60D and D78Y). Additionally, Dr. Kotlikoff introduced the CaM mutation M144I for improved Ca2+ binding affinity (lowers the Kd from 235 nM to 188 nM) - resulting in the GCaMP8.1 variant.
The fluorescent protein mVermilion is a derivative of mCherry with mutations giving it approximately 2-fold brighter emission.
|Allele Name||transgene insertion B34-4, Michael I Kotlikoff|
|Allele Type||Transgenic (Reporter)|
|Gene Symbol and Name||Tg(Acta2-GCaMP8.1/mVermilion)B34-4Mik, transgene insertion B34-4, Michael I Kotlikoff|
|Strain of Origin||C57BL/6J x SJL/J|
|Molecular Note||Acta2-GCaMP8.1-mVermilion transgenic mice (also called Acta2-GCaMP8.1/mVermilion or Acta2 |
Mice hemizygous for the Acta2-GCaMP8.1-mVermilion transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. When maintaining our live colony, hemizygous mice may be bred to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice (Stock No. 000664).
To date (July 2019), it has not been attempted to make this strain homozygous.
When using the Acta2-GCaMP8.1-mVermilion , Acta2BAC-GCaMP8.1-mVermilion , Acta2-GCaMP8.1/mVermilion mouse strain in a publication, please cite the originating article(s) and include JAX stock #032887 in your Materials and Methods section.
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