Important Note: The CAG-LSL-Gq-DREADD allele (formerly called R26-LSL-Gq-DREADD) was originally designed as a targeted insertion into the Gt(ROSA)26Sor locus. In 2017 The Jackson Laboratory confirmed the Rosa-CAG-LSL-HA-hM3Dq-pta-mCitrine construct randomly integrated into the mouse genome as a transgene, rather than at the Gt(ROSA)26Sor locus. To the best of our knowledge based on peer reviewed literature published to date (March 2017), the random integration event has not adversely affected the functionality of the allele.

The CAG-LSL-Gq-DREADD mice (formerly called R26-LSL-Gq-DREADD) have the Rosa-CAG-LSL-HA-hM3Dq-pta-mCitrine conditional allele (R26-hM3Dq/mCitrine). The CAG promoter-driven, loxP-flanked STOP cassette and HA-hM3Dq-pta-mCitrine coding region is designed to allow HA-hM3Dq-pta-mCitrine expression to be determined by which tissue(s) express Cre recombinase. Cre recombinase-mediated removal of the floxed-STOP cassette results in expression of two proteins; the yellow-green fluorescent protein mCitrine and the hemagglutinin epitope-tagged, mutant G protein-coupled receptor hM3Dq (a human muscarinic 3 receptor with two amino acid substitutions [Y149C^3.33/A239G^5.46] 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.

The donating investigator reports that CAG-LSL-Gq-DREADD mice do not express HA-hM3Dq or mCitrine prior to introduction of Cre recombinase. Following Cre recombinase exposure, mCitrine expression is detected by fluorescence (in the cytoplasm) and by immunohistochemical staining against the HA tag (in the membrane). The donating investigator has not observed canonical G_q pathway induction prior to the administration of CNO. CAG-LSL-Gq-DREADD hemizygous mice are viable and fertile, with no reported phenotypic abnormalities. While homozygous mice are expected to be viable and fertile, the generation of homozygous mice has not been attempted to date (January 2015).

Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired. Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired.

These CAG-LSL-Gq-DREADD mice may be useful for chemogenetic/pharmacogenetic studies of receptor-specific functions or general downstream cellular signaling emanating from the activated G protein-coupled receptor (GPCR). For example, when bred to a strain expressing Cre recombinase in forebrain neurons (Camk2a-Cre; Stock No. 005359), the resulting CaMKIIα-hM3Dq double mutant mice are a model allowing in vivo chemical control of in vivo chemical control of neuronal activity, neuronal firing, and non-neuronal signaling.

For CNO protocol, see the detailed protocol for dissolving CNO obtained from the attachments section of the Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.

Fixatives (formalin, paraformaldehyde and glutaraldehyde) may alter tissues, such as brain tissue, and induce autofluorescence in this and other fluorescent protein strains. A protocol to reduce autofluorescence in fixed brain tissue is available here.

Additional information on hM3Dq used in these CAG-LSL-Gq-DREADD mice:
The hM3Dq sequence is a G_q-coupled human M3 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the hM3Dq sequence, the wildtype human muscarinic 3 receptor (CHRM3) sequence was modified via site-directed mutagenesis to harbor two amino acid substitutions (Y149C^3.33/A239G^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).
More information on hM3Dq or other DREADDs may be available at Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.

Additional information on mCitrine used in these CAG-LSL-Gq-DREADD mice:
mCitrine is a yellow-green variant of Citrine YFP additionally harboring the A206K mutation. mCitrine is a ~5.7 pKa monomer with 516nm excitation and 529nm emmission spectra. Compared to wildtype GFP, Citrine harbors the S65G, V68L, Q69M, S72A and T203Y mutations, and mCitrine additionally has the A206K mutation (enhancing its ability to be a stable monomer).

Of note, several chemogenetic/pharmacogenetic tool strains are available from The Jackson Laboratory Repository; including these Cre-inducible CAG-LSL-Gq-DREADD mice (Tg-hM3Dq/mCitrine; Stock No. 026220), the Cre-inducible R26-LSL-Gi-DREADD mice (R26-hM4Di/mCitrine; Stock No. 026219), the Tet-responsive TRE-hM4Di transgenic mice (Stock No. 024114), the Tet-responsive TRE-hM3Dq transgenic mice (Stock No. 014093) and the adora2A-rM3Ds transgenic mice (Stock No. 017863).