Expression of "the HA-tagged hM4D pharmacologic inhibition tool" - a mutant human cholinergic receptor muscarinic 4 (CHRM4) that is responsive only to the ordinarily biologically inactive compound, clozapine-N-oxide (CNO) - and of a farnesylated red fluorescent protein is driven by the promoter of the mouse nuclear receptor subfamily 5, group A, member 1 (Nr5a1) gene. The transgene was made by replacing the translation start codon of the mouse Nr5a1 gene in the bacterial artificial chromosome (BAC) RP23-225F with a construct containing a cDNA encoding N-terminally hemagglutinin- (HA-) tagged CHRM4 with non-conservative substitutions for two strictly conserved amino acids (Tyr113Cys and Ala203Gly)("hM4D"); a viral A2 sequence to allow translation of two independent proteins from a single open reading frame; the coding sequence for the tdTomato fluorescent protein with a C-terminal farnesylation domain; a frt-flanked neomycin/kanamycin resistance cassette; and a polyadenyltion signal. Reporter protein fluorescence is observed in the dorsomedial and central but not the ventrolateral region of the ventromedial hypothalamus (VMH) and in the supraoptic commissure and dorsal periaqueductal gray area. Double immunofluorescence staining using primary antibodies to NR5A1 and HA demonstrates high-level expression of the transgene specifically in endogenously NR5A1-expressing cells. (J:207768, J:235494, J:235495)
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