Tg(Adora2a-Chrm3*,-mCherry)AD6Blr

The RP24-238K3 BAC harboring the adenosine A2a receptor locus (Adora2a), a ~40 kbp sequence proximal to the Adora2a locus (including part of the Specc1l locus) and ~117 kbp of sequence distal to the Adora2a locus (including the Upb1 locus and most of the 1110038D17Rik locus). A construct containing the mutant G protein-coupled receptor (GPCR) sequence, rM3Ds, with N-terminal hemagglutinin tag, an internal ribosome entry site (IRES), and mCherry fluorescent protein cDNA sequence was inserted downstream of the ATG start site of the Adora2a locus on the BAC via homologous recombination/BAC recombineering. The rM3Ds sequence is a Galphas-coupled rat muscarinic 3 DREADD (designer receptor exclusively activated by designer drug), also referred to as R-s, rM3/turkey beta1AR chimer DREADD, Gs-DREADD, Gs-D, or D-s. To create the rM3Ds sequence, the wild-type rat muscarinic 3 receptor (Chrm3) 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). Line AD6 was generated. (J:191042)