The ~184 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-370F21 contains 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 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 Acta2 gene (replacing the initiation codon of Acta2 in exon 2). No other loci on the BAC were altered. Mice from founder line 1 exhibit high sensor GCaMP-GR expression (mCherry fluorescence before calcium and EGFP fluorescence after calcium) in smooth muscle actin, including vessels. Transgene expression is consistent with endogenous Acta2. 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. (J:225491)
中文
