HCN4-GCaMP8 transgenic mice were designed in the laboratory of Dr. Michael I. Kotlikoff (Cornell University) as part of Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).
The ~237 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-281H22 was obtained; 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). Using homologous recombination/BAC recombineering, a GCaMP8-pA construct (GCaMP8 followed by an SV40 polyadenylation signal and flanking vector sequence) was inserted 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 resulting ~247 kbp modified BAC (Hcn4BAC-GCaMP8-pA) was purified and then microinjected into the male pronucleus of FVB x B6(Cg)-Tyrc-2J/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Hcn4BAC-GCaMP8 founder line B10-3 (R24:B10:L3) was identified with high sensor expression in heart (SA and AV nodes) and nervous system (brain and spinal cord). The donating investigator reports that HCN4-GCaMP8 transgenic mice from this founder line were bred to C57BL/6 for a total of two generations, and then black hemizygous mice were sent to The Jackson Laboratory Repository in 2016. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a C57BL/6 inbred male (thus the Y chromosome of the congenic strain is of C57BL/6 origin).
The genetically encoded calcium indicator GCaMP8 is a sensitive GCaMP variant with high dynamic range and fast response kinetics (Ohkura et al. 2012 PLoS One 7:e51286). GCaMP8 is composed of (from N-term to C-term) a polyHis plasmid leader sequence essential for thermal stability (RSET with the ΔH and ΔR2 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).
