Tg(Gja5-GCaMP2)1Mik

Mice bearing this transgene express the Ca2+ sensor GCaMP2 in atrial myocytes, the Purkinje cell network and endothelial cells lining cardiac arterioles, as well as in endothelium of arteries, arterioles and capillaries of various non-cardiac tissues, under control of the regulatory sequences of gap junction membrane channel protein alpha 5 (Gja5/Cx40). The transgene was created from bacterial artificial chromosome (BAC) clone RP24-244O4, containing the C57BL/6J mouse Gja5 gene and approximately 85 kb of 5' and 61 kb of 3' flanking genomic DNA, by replacing the first two codons of Gja5, in exon 2, with two copies of GCaMP2 separated by an internal ribosome entry site (IRES) and followed by a polyadenylation signal (GCaMP2-IRES-GCaMP2-pA). GCaMP2 is a Ca2+-sensing protein consisting of a circularly permuted EGFP, with the positions of the N-terminal 144 amino acids (aa) and the C-terminal 90 aa reversed, and with a 13-aa peptide of myosin light chain kinase (M13) and calmodulin (CaM) appended to the new N- and C-termini, respectively. The 35-aa polyHis plasmid leader sequence RSET, found to be essential for thermal stability, resides N-terminal to M13, and mutations have been introduced to improve fluorescence (Asp180Tyr and Val93Ile) and thermal stability (Val163Aln and Ser175Gly) and to prevent GFP dimerization (Ala206Lys). Fluorescence of GCaMP2 is induced by interaction of the C-terminal calmodulin, in association with Ca2+, with the N-terminal M13. (J:107637, J:125615)