R26-LSL-Gi-DREADD mice have the Rosa-CAG-LSL-HA-hM4Di-pta-mCitrine conditional allele (R26-hM4Di/mCitrine) that has a loxP-flanked STOP cassette designed to prevent transcription of the downstream HA-hM4Di-pta-mCitrine coding region - although some expression may be observed before Cre recombinase (see below). Because the CAG promoter-driven reporter construct is inserted into the Gt(ROSA)26Sor locus, higher levels of HA-hM4Di and mCitrine expression are expected in the tissue(s) expressing Cre recombinase.

Importantly, several investigators have reported expression (mCitrine fluorescence) prior to introduction of Cre recombinase [2019]. The mCitrine fluorescence after Cre recombination is expected to be significantly greater than those baseline levels. Because of the expression before Cre, this strain may not be appropriate for single-cell applications in which the reporter is necessary for the identification of DREADD-expressing cells. Furthermore, it is recommended that researchers include Cre-negative R26-LSL-Gi-DREADD controls to establish the baseline expression levels in their experiments.

Cre recombinase-mediated removal of the floxed-STOP cassette can result in robust expression of two proteins: the yellow fluorescent protein variant mCitrine and the hemagglutinin epitope-tagged, mutant G protein-coupled receptor hM4Di (a human muscarinic 4 receptor with two amino acid substitutions [Y113C^3.33/A203G^5.46] that abolishes receptor affinity for the native ligand, acetylcholine [ACh], but allows receptor binding and subsequent activation by the small pharmacologically inert molecule clozapine-N-oxide [CNO]). hM4Di activation via CNO binding induces the canonical G_i pathway - leading to neuronal silencing.

In 2015, the donating investigator reported that 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_i pathway induction prior to the administration of CNO.

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 R26-LSL-Gi-DREADD mice may be useful for chemogenetic/pharmacogenetic studies of receptor-specific functions or general downstream cellular signaling emanating from activation of an inhibitory 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-hM4Di double mutant mice are a model allowing in vivo chemical control of membrane hyperpolarization and neuronal silencing.

R26-LSL-Gi-DREADD heterozygous 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].

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.

Of note, several chemogenetic/pharmacogenetic tool strains are available from The Jackson Laboratory Repository; including these Cre-inducible R26-LSL-Gi-DREADD mice (R26-hM4Di/mCitrine; Stock No. 026219), the Cre-inducible CAG-LSL-Gq-DREADD mice (Tg-hM3Dq/mCitrine [formerly R26-LSL-Gq-DREADD]; Stock No. 026220), 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).