Tg(Cdh5-GCaMP8)B20-6Mik

The ~200 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-453P1 was obtained; containing the entire cadherin 5 locus (Cdh5) as well as ~124 kbp of 5' flanking sequences (including the complete Gm32728, Gm39236 and Gm8748 predicted gene loci) and ~33 kbp of 3' flanking sequences (including a 5' portion of the Bean1 locus). Using homologous recombination/BAC recombineering, a 2230 bp GCaMP8-SV40pA construct was inserted into the ATG start site of the BAC Cdh5 gene (replacing the initiation codon of Cdh5). No other loci on the BAC were altered. The 11.6 kbp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette. GCaMP8 is composed of (from N-term to C-term) a polyHis plasmid leader sequence essential for thermal stability, a 20 residue peptide of chicken smooth muscle myosin light chain kinase (M13; target peptide for a Ca2+-bound CaM), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144), a rat calmodulin DNA fragment (CaM; aa 2-148). Founder line B20-6 was identified with high sensor expression in vascular endothelial cells. (J:314125)