Gt(ROSA)26Sor^{tm3(CAG-Chrm3*,-mCitrine)Ute}

The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a hemagglutinin epitope tag (HA), the rM3Ds sequence, a porcine teschovirus (PTA) cleavage site, the mCitrine fluorescent protein, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor. The rM3Ds sequence is a Galpha>s<-coupled rat muscarinic 3 DREADD (designer receptor exclusively activated by designer drug). To create the rM3Ds sequence, the wildtype 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). mCitrine is a yellow-green variant of Citrine YFP additionally harboring the A206K mutation. mCitrine is a ~5.7 pKa monomer with 516nm excitation and 529nm emmission spectra. Compared to wildtype GFP, Citrine harbors the S65G, V68L, Q69M, S72A and T203Y mutations, and mCitrine additionally has the A206K mutation (enhancing its ability to be a stable monomer). (J:101977)