B6.Cg-Tg(Gja5-GCaMP3*/mCherry)B15-2Mik/J

Stock number: 030333
Product name: Cx40-GCaMP-GR , Cx40^BAC-GCaMP-GR , Cx40^BAC-GCaMP-GR
Research areas: Cardiovascular Research, Cell Biology Research, Internal/Organ Research, Neurobiology Research, Research Tools

Description

Cx40-GCaMP-GR transgenic mice (cx40^BAC-GR) are a fluorescent calcium indicator strain that exhibits high/robust calcium-sensor expression in arterial endothelial cells, atrial myocytes and cardiac Purkinje fibers. The ratiometric GCaMP-GR allows calcium-independent bright mCherry fluorescence when GCaMP fluorescence is dim (before calcium transients). Upon calcium binding, this pore becomes occupied and EGFP fluorescence is increased.

Detailed description

Cx40-GCaMP-GR transgenic mice (cx40^BAC-GR) express the ratiometric fluorescent calcium indicator GCaMP-green/red (GCaMP-GR) under control of the Gja5 locus (connexin40 or Cx40) promoter/enhancer regions within the BAC transgene. Cx40-GCaMP-GR mice from founder line B15-2 exhibit high/robust sensor expression (constant mCherry fluorescence and increased EGFP fluorescence after calcium) in arterial endothelial cells, atrial myocytes and cardiac Purkinje fibers. Expression is also observed in endothelium of arteries, arterioles and/or capillaries of various non-cardiac tissues (including lung, liver, aorta and skeletal muscle). No expression is reported in ventricular myocardium or venous endothelial cells (lung or skeletal muscle). The donating investigator reports that, in total, the Cx40-GCaMP-GR transgene expression pattern has high fidelity with that of endogenous Gja5.

Cx40-GCaMP-GR may be useful for studying the effect of histone proteins on endothelial cell Ca2+ signaling [Collier et al. 2019 Am J Physiol Heart Circ Physiol (PMID:30848676)].

The GCaMP-GR is designed to exhibit bright mCherry fluorescence and low/baseline EGFP fluorescence in the absence of calcium binding, and then following calcium binding (such as arteriolar vasodilation), increased EGFP fluorescence while mCherry expression remains constant. Additional GCaMP-GR information is described further below.

Mice hemizygous for the Cx40-GCaMP-GR transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. To date (February 2019), it has not been attempted to make this strain homozygous.

Calcium is a key molecular signal for cell functions including heart, vessel, and airway contraction, lung secretion, autonomic neurotransmission and immunocyte function. Cx40-GCaMP-GR transgenic mice may be useful to examine calcium signaling in endothelium-dependent vasodilation both in vivo and in vitro.

The ratiometric calcium indicator GCaMP-GR (green/red) is an optimized GCaMP3 variant (A206V, S30R, Y39N and N105T mutations in EGFP) fused to the mCherry fluorescent protein by a flexible alanine-proline repeat linker (GSSTSG-APAPAPAPAPAPAP-SEF) designed by Dr. Michael I. Kotlikoff (Cornell University; Shui et al. 2014 Front Physiol 5:428). Compared to GCaMP3, GCaMP-GR has equivalent calcium sensitivity, lower resting fluorescence, higher dynamic range and calcium-independent mCherry fluorescence. GCaMP-GR allows calcium-independent bright mCherry fluorescence before calcium transients when GCaMP fluorescence is dim (as GCaMP has a pore from the outside of its barrel into the chromophore). Upon calcium binding, this pore becomes occupied and EGFP fluorescence is increased.

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.

Development

Cx40-GCaMP-GR transgenic mice (also called cx40^BAC-GR or cx40^BAC-GCaMP-GR) 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 ~167 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP24-255O4 was obtained; containing the entire Gja5 locus (connexin40 or Cx40) as well as ~85 kbp of 5' flanking sequences and ~61 kbp of 3' flanking sequences (including the partially overlapping 4930573H18Rik locus). Using homologous recombination/BAC recombineering, a 3006 bp GCaMP-GR-pA construct (GCaMP-GR [described below] followed by an SV40 polyadenylation signal and flanking vector sequence) was inserted into the ATG start site of the BAC Gja5 gene (replacing nucleotides aagatggg at the initiation codon of Gja5 in exon 2). No other loci on the BAC were altered. The 13.5 kbp BAC vector (pTARBAC1) contained a chloramphenicol selection cassette.
The resulting ~183 kbp modified BAC (cx40^BAC-GR) was purified and then microinjected into B6SJLF1/J x B6SJLF1/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Cx40-GCaMP-GR founder line B15-2 was identified with high sensor expression in arterial endothelial cells, atrial myocytes and cardiac Purkinje fibers. The donating investigator reports that Cx40-GCaMP-GR transgenic mice from this founder line were backcrossed to C57BL/6J for a total of at least 11 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 (February 2019).

The genetically encoded calcium indicator GCaMP-green/red (GCaMP-GR) is a calcium-sensing molecule composed of a 34 aa polyHis plasmid leader sequence (RSET with second amino acid arginine deleted; essential for thermal stability), a 13 residue peptide of chicken smooth muscle myosin light chain kinase (M13), 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 the N60D mutation to increase fluorescence change for small calcium transients]), a flexible alanine-proline repeat linker (GSSTSG-APAPAPAPAPAPAP-SEF) and the mCherry fluorescent protein. The cpEGFP mutations result in increased brightness (D180Y and V93I), thermal stability (V163A and S175G), dimerization prevention (A206K), dynamic range/baseline fluorescence (M153K and T203V) and enhanced folding activity/increased fluorescence (A206V, S30R, Y39N and N105T). The alanine-proline repeat linker was optimized to maintain full GCaMP dynamic range as well as mCherry brightness. [Shui et al. 2014 Front Physiol 5:428]

Allele

Symbol: Tg(Gja5-GCaMP3*/mCherry)B15-2Mik
Name: transgene insertion B15-2, Michael I Kotlikoff
MGI accession ID: MGI:6281630
Generation method: Transgenic
Attributes: Reporter; Inserted expressed sequence
Marker symbol: Tg(Gja5-GCaMP3*/mCherry)B15-2Mik
Marker name: transgene insertion B15-2, M I Kotlikof
Chromosome: UN
Strain of origin: (C57BL/6J x SJL/J)F2
Molecular note: The transgenic construct is made using a C57BL/6J mouse bacterial artificial chromosome (BAC) RP24-255O4 containing the entire Gja5 locus as well as ~85 kbp of 5' flanking sequences and ~61 kbp of 3' flanking sequences (including the partially overlapping 4930573H18Rik locus). Using homologous recombination/BAC recombineering, a 3006 bp GCaMP-GR-pA construct is inserted into the ATG start site of the BAC Gja5 gene (replacing nucleotides aagatggg at the initiation codon of Gja5 in exon 2). No other loci on the BAC were altered. The 13.5 kbp BAC vector (pTARBAC1) contained a chloramphenicol selection cassette. GCaMP-GR is a calcium-sensing molecule composed of a 34 aa polyHis plasmid leader sequence, a 13 residue peptide of chicken smooth muscle myosin light chain kinase, a circularly permutated EGFP, a rat calmodulin DNA fragment, a flexible alanine-proline repeat linker and the mCherry fluorescent protein. Founder line B15-2 has high sensor expression in arterial endothelial cells, atrial myocytes and cardiac Purkinje fibers.