Gt(ROSA)26Sor^{tm9.1(CAG-mCherry,-CHRM4*)Dym}

The targeting construct introduced into the locus a CAG promoter, FRT-flanked STOP cassette, loxP site, mCherry reporter gene, STOP cassette, loxP site and hM4Di sequence is a Gi-coupled human M4 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the hM4Di sequence, the wildtype 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). Flp-mediated recombination removed the first STOP cassette allowing for expression of the mCherry reporter gene. Cre-mediated recombination is required to achieve expression of the hM4Di sequence. (J:234693)