B6.129-Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute}/J

Stock number: 026219
Product name: R26-LSL-Gi-DREADD , R26-LSL-hM4Di-DREADD
Strain synonyms: R26-hM4Di/mCitrine
Research areas: Cancer Research, Cell Biology Research, Diabetes and Obesity Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

R26-LSL-Gi-DREADD mice were designed to have Cre recombinase-inducible expression of a CAG promoter-driven HA-hM4Di-pta-mCitrine from the Gt(ROSA)26Sor locus. Following Cre-mediated removal of an upstream floxed-STOP cassette, higher expression levels of HA-tagged hM4Di and mCitrine yellow fluorescent protein is observed. See Important Note regarding expression levels before Cre.

hM4Di is a mutant G protein-coupled receptor (GPCR) that induces the canonical G_i pathway specifically following administration of the pharmacologically inert molecule clozapine-N-oxide (CNO). These R26-LSL-Gi-DREADD mice may be useful for chemogenetic/pharmacogenetic studies to express an inhibitory DREADD that effectively silences the activity of neurons.

Detailed description

Important Note: Several investigators using R26-LSL-Gi-DREADD mice 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.

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.

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. Additionally, The Jackson Laboratory finds that homozygous mice are viable and fertile [April 2020].

HA Expression Before and After Cre ; SOP for Brain Immunofluorescence: In 2022, The Jackson Laboratory bred some R26-LSL-Gi-DREADD homozygous females with Sox2-Cre hemizygous males (Stock No. 008454). The resulting offspring were used to examine HA-hM4Di expression via HA antibody immunofluorescent imaging. The Cre-negative R26-LSL-Gi-DREADD heterozygous offspring had weak occasional HA staining, while the Cre-positive R26-LSL-Gi-DREADD heterozygous offspring showed positive HA staining throughout brain that was brighter than Cre-negative littermates and much brighter than the 026219 parental samples. Furthermore, we recommend immunofluorescent imaging rather than western blot to detect HA. Our results and our SOP for examining brain immunofluorescence is available here: 026219 HA expression before Cre and after Cre ; SOP Brain Immunofluorescence 026219 026220.

Protocol to reduce autofluorescence in fixed brain tissue: 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 [Oct 2017].

In addition to these Cre-inducible R26-LSL-Gi-DREADD mice (R26-hM4Di/mCitrine; Stock No. 026219), a Cre-inducible Gq-DREADD mouse line is also available (Tg-hM3Dq/mCitrine [formerly R26-LSL-Gq-DREADD]; Stock No. 026220).

See the JAXMice search page for chemogenetic/pharmacogenetic tool strains available from The Jackson Laboratory.

Development

The R26-LSL-Gi-DREADD mice were created at Duke University by Drs. Ute Hochgeschwender (while at Duke University) and Bryan L. Roth (University of North Carolina Chapel Hill Medical School). The Rosa-CAG-LSL-HA-hM4Di-pta-mCitrine targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a frt site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a hemagglutinin epitope tag (HA), the hM4Di sequence (described in detail below), a porcine teschovirus (PTA) cleavage site, the mCitrine fluorescent protein (described in detail below), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-frt-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred with C57BL/6NCrl mice. Heterozygotes were further crossed to C57BL/6NCrl for several generations by the donating lab (see SNP notes below). In 2015, heterozygous males from generation N6 were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To generate the living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ female mice (Stock No. 005304). The R26-LSL-Gi-DREADD mice were then bred together, to wildtype mice from the colony or to C57BL/6NJ inbred animals. The donating investigator reports they bred a R26-LSL-Gi-DREADD female to a C57BL/6N male (thus the Y chromosome is from the C57BL/6N background).

The hM4Di sequence is a G_i-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 (Y113C^3.33/A203G^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).

Additional information on hM4Di:
More information on hM4Di or other DREADDs may be available at Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.

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).

In 2015, a 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on a cohort of the first generation rederived mice [generation N6+pN1] at The Jackson Laboratory. This found all markers were C57BL/6N allele-type.

In 2017, our 48 SNP panel was expanded to have 43 markers throughout the genome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains. Testing a cohort of mice from The Jackson Laboratory living colony [generations N6+pN1F5 and N6+pN1F6] found some of the mice had 2 SNPs segregating. The first marker was one of the new C57BL/6N vs. C57BL/6J substrain SNPs at Chromosome 6 ~108Mb. This marker is only ~5Mb away from the targeted Gt(ROSA)26Sor locus, and over time our experience has been that Gt(ROSA)26Sor mutant strains often retain targeted ES cells genomic contributions at this SNP. Furthermore, this Chromosome 6 marker is also known to have the same SNP for C57BL/6J and C57BL/6NCrl (the original background of the donated strain). As such, this Chromosome 6 marker is considered uninformative for determining this strains congenicity. The second marker was one of the new genomic SNPs at Chromosome 7 ~36Mb showing C57BL/6N and 129 allele-types. The closest marker to this on the previous SNP panel was at Chromosome 7 ~4Mb which showed as C57BL/6N allele-type. Taken all together, these data support the donors' report that the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.

Of note by 2023, The Jackson Laboratory live colony [generations N6+pN3F9, N6+pN3F10 and N6+pN3F11] SNP panel results showed all tested cohorts as C57BL/6N-allele type at the Chromosome 7 ~36Mb marker.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute}
Name: targeted mutation 1, Ute Hochgeschwender
MGI accession ID: MGI:5614050
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: hM4Di; LSL-hM4Di; R26^hM4D; R26-hM4Di/mCitrine; R26-LSL-Gi-DREADD; R26-LSL-hM4Di/mCitrine
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: CHRM4,cholinergic receptor muscarinic 4,human; YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: Yellow fluorescent protein variant mCitrine and the hemagglutinin epitope-tagged, mutant G protein-coupled receptor hM4Di (modified CHRM4 sequence) will be expressed in tissues expressing Cre recombinase.
Molecular note: The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a hemagglutinin epitope tag (HA), the hM4Di sequence, a porcine teschovirus (PTA) cleavage site, the mCitrine fluorescent protein, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.

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 (Y113C<3.33>/A203G<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).

mCitrine is a yellow-green variant of Citrine YFP additionally harboring the A206K mutation. mCitrine is a approx 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).