Cacna1h^{tm1.1(epH)Ebou}

The Cacna1h (also known as Cav3.2) gene was modified by the insertion of the DNA sequence encoding "ecliptic pHlourin" (also called "ecliptic GFP"; a pH-sensitive derivative of wild-type green fluorescent protein), in-frame, into exon 6 and of loxP sites upstream of exon 6 and downstream of exon 7; a frt-flanked neomycin selection cassette upstream of the first loxP site was excised by flp recombinase. In the resultant fusion protein, ecliptic GFP is part of a large extracellular loop preceding the P loop of the first transmembrane domain. Northern blot and in-situ hybridization analyses with Cacna1h and GFP probes and western blot and fluorescence immunohistology analyses with antibodies to GFP and CACNA1H verified expression of the fusion mRNA and protein, respectively, in whole brain, whole dorsal root ganglion (DRG) and DRG sections from homozygous mutant adult mice. Functionality of the fusion protein-derived channels is demonstrated by electrophysiological analysis of live DRG tissue from homozygous mutant mice. Crossing mice with this allele to cre recombinase-expressing mice results in excision of exons 6 and 7 (and the GFP insertion) and production of a non-functional calcium channel. (J:94746, J:220891)