Gt(ROSA)26Sor^{tm3(CAG-CHRM4*,-tdTomato)Ghsh}

A targeting vector, which contained the CAG promoter, a loxP site flanked STOP fragment, an FRT site flanked STOP fragment, the human muscarinic acetylcholine receptor subtype 4 DREADD (hM4D) and tdTomato, was inserted into the locus via homologous recombination. The hM4D sequence is a G-coupled human M4 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the hM4D sequence, the wild-type human muscarinic 4 receptor (CHRM4) sequence was modified via site-directed mutagenesis to harbor two amino acid substitutions (Y113C<3.33>/A203G<5.46>) 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:226610)