Tg(Hcn4-GCaMP8)B10-3Mik

The ~237 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-281H22 containing the entire hyperpolarization-activated, cyclic nucleotide-gated K+ 4 locus (Hcn4), as well as ~66 kbp centromeric flanking sequences and ~134 kbp telomeric flanking sequences (including a portion of the Neo1 locus) was modified by inserting the GCaMP8-pA construct (GCaMP8 followed by an SV40 polyadenylation signal and flanking vector sequence) into the ATG start site of the BAC Hcn4 gene (replacing the initiation codon of Hcn4 in exon 1). No other loci on the BAC were altered. The genetically encoded calcium indicator GCaMP8 is a sensitive GCaMP variant with high dynamic range and fast response kinetics. GCaMP8 is composed of (from N-term to C-term) a polyHis plasmid leader sequence essential for thermal stability (RSET with the DeltaH and DeltaR2 mutations), a 13 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 [with mutations listed below]), a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations listed below]). The cpEGFP mutations result in improved brightness (D180Y and V93I), thermal stability (V163A and S175G), dimerization prevention (A206K), dynamic range/baseline fluorescence (M153K, T203V, N105Y and E124V), and overall functionality (S205N and I47F). The CaM mutations are for higher calcium affinity (M36L), increased fluorescence change for small calcium transients (N60D) and chromophore stabilization (D78Y). (J:314125)