Gt(ROSA)26Sor^{tm2(CAG-mGCaMP3)Dbe}

The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP site, an FRT-flanked neomycin resistance cassette in reverse orientation to the gene, a STOP cassette (3x SV40 polyA), a second loxP site, a GCaMP3 fusion gene, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability) followed by a BGH polyA signal. The GCaMP3 fusion gene contains a cDNA sequence encoding the chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (amino acid residues 149-238 followed by amino acid residues 1-144 (including M153K, V163A, S175G, D180Y, T203V, A206K, and V93I mutations to increase dynamic range and baseline fluorescence), and a DNA fragment encoding amino acid residues 2-148 of the rat calmodulin (modified to harbor the N60D mutation to increase the fluorescence change for small calcium transients). To localize GCaMP3 to the plasma membrane, a gene sequence encoding the first 8 amino acids of the modified Marcks (myristoylated alanine rich protein kinase C substrate) sequence (MGCCFSKT) was fused to the transcription initiation codon. The targeting vector is inserted between exons 1 and 2. (J:254093)