Tg(tetO-CHRM4*)2Blr

The transgenic construct includes a modified Tet response element (TRE or (tetO), a hemagglutinin epitope tag (HA), the hM4Di sequence and an SV40 polyA signal. The 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 (Y113C3.33/A203G5.46) 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). The TRE promoter is the Tet-responsive Ptight promoter. This Ptight promoter contains a modified Tet response element (TREmod), which consists of seven direct repeats of a 36-bp sequence each containing the 19-bp tet operator sequence (tetO). The TREmod is just upstream of a minimal human cytomegalovirus (CMV) promoter (PminCMVDelta), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P,tight is silent in the absence of binding of TetR or rTetR to the tetO sequences.