Tg(Ins2-Chrm3*)SJwe

The transgene was designed to have (from 5' to 3') a 650 bp rat insulin 2 (Ins2; RIP II) promoter fragment, a hemagglutinin epitope tag (HA), the GsD coding sequence (R-s; described below), and untranslated human growth hormone sequences (containing transcriptional termination, polyadenylation and splicing signals). The GsD sequence is a Galpha>s<-coupled rat M3 DREADD (designer receptor exclusively activated by designer drug). To create the rM3Ds sequence, the wildtype rat muscarinic 3 receptor (Chrm3; M3R) sequence was first modified to replace the second and third intracellular loops with the corresponding turkey beta1-adrenergic receptor (ADRB1 or beta1AR) sequences. The second modification introduced, via site-directed mutagenesis, the Y148C and A238G amino acid substitutions that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small drug-like molecule clozapine-N-oxide (CNO). (J:154657)