The ~184 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-370F21 was 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 GCaMP2-pA construct (GCaMP2 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 6 exhibit high sensor expression (GCaMP2 expression/EGFP fluorescence) in smooth muscle actin, including vessels. Transgene expression is consistent with endogenous Acta2. The genetically encoded calcium indicator GCaMP2 is a calcium-sensing molecule composed of a 35 aa polyHis plasmid leader sequence (RSET; 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]), and a rat calmodulin DNA fragment (CaM; aa 2-148). The cpEGFP mutations result in increased brightness (D180Y and V93I), thermal stability (V163A and S175G) and GFP dimerization prevention (A206K). (J:314125)
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