Tg(RP23-370F21-RCaMP1.07)B3-3Mik

The ~175 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-370F21 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)was modified by insertion a RCaMP1.07-pA construct (RCaMP1.07 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. The genetically encoded calcium indicator RCaMP1.07 is a red calcium-sensing molecule that functions at cellular calcium levels, and is a modified version of R-CaMP1.01 (which was created by insertion of random mutations into R-GECO1). RCaMP1.07 is composed of (from N-term to C-term) 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; target peptide for a Ca2+-bound CaM), a circularly permutated red fluorescent protein (mApple; aa 146-231 followed by aa 1-145 [with mutations described below]), a rat calmodulin DNA fragment (CaM; aa 2-148), a 15 aa linker and a self-cleaving peptide (F2A). Compared to R-GECO1, the mApple mutations K47V and T49V result in increased Ca2+-dependent fluorescent change. The C-terminal addition of F2A also increases the Ca2+-dependent fluorescent change, as well as exports RCaMP1.07 out of the nucleus. (J:314125)