Tg(Hcn4-COP4*L132C,-lacZ)B17-2Mik

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 3' portion of the Neo1 locus). Using homologous recombination/BAC recombineering, a 5640 bp CatCh-IRES-lacZ-pA construct 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 11.6 kbp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette. The calcium translocating channelrhodopsin CatCh [ChR2(L132C)] cDNA sequence used here encodes the first 309 amino acids of green alga Chlamydomonas reinhardtii channelrhodopsin-2 (ChR2 or cop4) that has been modified with an L132C amino acid substitution (CTC-to-TGC) designed to cause enhanced Ca2+ permeability. Founder line B17-2 has high effector and lacZ expression in heart (SA and AV nodes). (J:314125)