RC::L-hM3Dq is a Cre recombinase-responsive Gq-coupled DREADD allele with a cre-dependent FLEx switch cassette containing an eGFP sequence and an inverted hM3Dq/mCherry fusion protein that are uniquely flanked/separated by inward-facing lox sites (both lox2272 and loxP).

Under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread and robust eGFP fluorescence is observed, but the inverted hM3Dq/mCherry sequences are not epxressed. Exposure to Cre recombinase that places hM3Dq/mCherry into the proper orientation next to the CAG promoter (see details below) results in expression of the hM3Dq-mCherry-2ACT88 fusion protein; a mutant G protein-coupled receptor hM3Dq fused to the red fluorescent protein mCherry, with a rat Htr2A C-terminal epitope (2ACT88) that mediates localization to the cell body/soma and dendrites.
hM3Dq is a G_q-coupled human M3 muscarinic DREADD (designer receptor exclusively activated by designer drug) with two amino acid substitutions that abolish receptor affinity for the native ligand, acetylcholine [ACh], but allow receptor binding and subsequent activation by the small pharmacologically inert molecule clozapine-N-oxide [CNO]). hM3Dq activation via CNO binding induces the canonical G_q pathway; leading to neuronal activity/neuronal firing.

Specifically, the donating investigator reports that mice homozygous for the RC::L-hM3Dq allele are viable and fertile with no reported gross physical or behavioral abnormalities. Prior to Cre recombinase exposure, RC::L-hM3Dq exhibit widespread eGFP fluorescence that is sufficiently robust to be detected by direct fluorescence in live or fixed tissue, as well as by immunohistochemistry. Before cre introduction, RC::L-hM3Dq show neither hM3Dq/mCherry fusion expression nor ectopic mCherry immunofluorescence in any of the tested tissues.


When RC::L-hM3Dq are bred to mice with germline expression of Cre recombinase (Tg(ACTB-cre)2Mrt ; see Stock No. 019099), the resulting offspring demonstrate ubiquitous expression of hM3Dq/mCherry in both embryos and adult brain. mCherry fluorescence is sufficiently robust to be detected by direct fluorescence in live or fixed tissue, as well as by immunohistochemistry.


Similarly, breeding RC::L-hM3Dq to have either the Camk2a-cre transgene (Stock No. 005359) or the GFAP-cre/ERT2 transgene (similar to Stock No. 012849) resulted in hM3Dq/mCherry expression in the soma/dendrites of neurons or tamoxifen-inducible hM3Dq/mCherry expression in the soma/dendrites of glia, respectively. Further administration of CNO evoked physiological signatures ascribed to activation of Camk2a+ neurons and GFAP+ glia, respectively.


To demonstrate intersectional Flp/Cre control, RC::FL-hM3Dq mice (Stock No. 026942)
were bred to be heterozygous for the Dbh^Flpo knockin/knockout allele and the En1^cre knockin/knockout allele (see Stock No. 007916). In the resulting triple heterozygous mice, hM3Dq/mCherry expression (mCherry fluorescence) was restricted to the soma and dendrites of a subpopulation of noradrenergic neurons (LC complex), whereas all remaining noradrenergic neurons (i.e. expressing only Dbh^Flpo) were labeled with eGFP. In vitro administration of CNO depolarized/activated the hM3Dq/mCherry+ LC complex neurons, while CNO had no effect on membrane potential of eGFP+ control neurons.

Importantly, several cre-mediated recombination outcomes of the RC::L-hM3Dq allele are possible based on which lox sites are recombined. The outcomes are described below, along with some experimental considerations:


  i. inversion of the RC::L-hM3Dq allele at the lox2272 sites places the hM3Dq/mCherry sequence adjacent to the CAG promoter and into the proper orientation for expression, with the downstream eGFP cassette inverted (and now flanked with loxP sites in the same orientation); resulting in red fluorescence and DREADD expression.

     i-i. inversion of outcome i at the lox2272 sites reverts to the original RC::L-hM3Dq allele; resulting in restored eGFP expression and no red fluorescence or DREADD expression.

     i-ii. recombination of outcome i at the loxP sites deletes the downstream eGFP cassette and one of the lox2272 sites; resulting in continued red fluorescence and DREADD expression that is now irreversible (independent of additional Cre).

  ii. inversion of the RC::L-hM3Dq allele at the loxP sites places the hM3Dq/mCherry into the proper orientation for expression, but the eGFP cassette (now flanked with lox2272 sites in the same orientation) remains adjacent to the CAG promoter; resulting in continued eGFP expression and no red fluorescence or DREADD expression.

     ii-i. inversion of outcome ii at the loxP sites reverts to the original RC::L-hM3Dq allele; resulting in continued eGFP expression and no red fluorescence or DREADD expression.

     ii-ii. recombination of outcome ii at the lox2272 sites deletes the eGFP cassette and one of the loxP sites; this places the properly-orientated hM3Dq/mCherry sequence adjacent to the CAG promoter and results in red fluorescence and DREADD expression that is now irreversible (independent of additional Cre).

The two lox variants, lox2272 and loxP, are compatible only with a lox sequence identical to self; they do not recombine with each other. Although some RC::L-hM3Dq recombination outcomes require more than one Cre recombinase pulse to achieve red/DREADD expression, no outcomes result in deletion of the hM3Dq/mCherry cassette. Because inward-facing lox sequences result in cre-mediated inversion rather than excision, only outcomes i-ii and ii-ii have irreversible red/DREADD expression; the other outcomes may be able to reversibly switch back-and-forth from the non-red/DREADD-expressing orientations as long as Cre recombinase is active in the cell. Those cells may eventually recombine to an irreversible red/DREADD-expressing orientation. To this point the donating investigator reports that, regardless of which inversion event occurs first, continuous exposure to Cre rapidly recombines the allele into the final state (irreversible red/DREADD-expression).