Overview

Also Known As:R26-LSL-Gi-DREADD , R26-LSL-hM4Di-DREADD, R26-hM4Di/mCitrine

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. 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.

Donating Investigator

Ute Hochgeschwender, Central Michigan University

Bryan L Roth, University of North Carolina at Chapel Hill

Genetic overview

Genetic Background	Generation
	N6+pN1F8 (2019-01-04 00:00:00)

Gt(ROSA)26Sor^{tm1(CAG-CHRM4,-mCitrine)Ute}*

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed), No functional change)	Gt(ROSA)26Sor	gene trap ROSA 26, Philippe Soriano

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Research Applications

Research Tools
Neurobiology Research
Immunology, Inflammation and Autoimmunity Research
Cancer Research
Diabetes and Obesity Research
Cell Biology Research

View All Research Applications

Base Price

Starting at:

$255.00 Domestic price for female 4-week

510.00 Domestic price for breeder pair

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Details

Detailed Description

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).

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. 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).

Expression Data

Expressed Gene	CHRM4, cholinergic receptor muscarinic 4, human
Expressed Gene	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.

Control Suggestions

Wild-type from the colony
005304 C57BL/6NJ

Additional Information

Considerations for Choosing Controls

Selected References

Zhu H; Aryal DK; Olsen RH; Urban DJ; Swearingen A; Forbes S; Roth BL; Hochgeschwender U. 2016. Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice. Genesis 54(8):439-46PubMed: 27194399 MGI: J:236327

View All References

Genetics

Gt(ROSA)26Sor^{tm1(CAG-CHRM4,-mCitrine)Ute}*

Allele Symbol: Gt(ROSA)26Sor^{tm1(CAG-CHRM4,-mCitrine)Ute}*

Allele Name	targeted mutation 1, Ute Hochgeschwender
Allele Type	Targeted (Conditional ready (e.g. floxed), No functional change)
Allele Synonym(s)	hM4Di; LSL-hM4Di; R26-hM4Di/mCitrine; R26-LSL-Gi-DREADD; R26-LSL-hM4Di/mCitrine
Gene Symbol and Name	Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano
Gene Synonym(s)
Promoter	CAG, CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter,
Expressed Gene	CHRM4, cholinergic receptor muscarinic 4, human
Expressed Gene	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.
Strain of Origin	(129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Chromosome	6
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).

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Research Tools
- Neurobiology Research
  - cell marker
- Fluorescent Proteins
- Cre-lox System
  - loxP-flanked Sequences: Test/Reporter
  - loxP-flanked Sequences
- Developmental Biology Research
  - Cre-lox System
- Genetics Research
  - Tissue/Cell Markers: Cre-lox System
Neurobiology Research
- Fluorescent protein expression in neural tissue
- Optogenetic and Chemogenetic tools
  - Chemogenetic tools
- Cre-lox System
  - loxP-flanked Sequences: Test/Reporter
  - loxP-flanked Sequences
- Receptor Defects
  - G protein-coupled receptor
Immunology, Inflammation and Autoimmunity Research
- Intracellular Signaling Molecules
- Growth Factors/Receptors/Cytokines
Cancer Research
- Growth Factors/Receptors/Cytokines
Diabetes and Obesity Research
- Insulin Receptors and Growth Factors
Cell Biology Research
- Signal Transduction

Mammalian Phenotype Terms by Genotype

The following phenotype relates to a compound genotype created using this strain

Genotype: Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute}/Gt(ROSA)26Sor⁺
B6N.129-Gt(ROSA)26Sor/J

normal phenotype

no abnormal phenotype detected
- mice are viable and fertile

References

Zhu H; Aryal DK; Olsen RH; Urban DJ; Swearingen A; Forbes S; Roth BL; Hochgeschwender U. 2016. Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice. Genesis 54(8):439-46PubMed: 27194399 MGI: J:236327

Additional References

Liu YU; Ying Y; Li Y; Eyo UB; Chen T; Zheng J; Umpierre AD; Zhu J; Bosco DB; Dong H; Wu LJ. 2019. Neuronal network activity controls microglial process surveillance in awake mice via norepinephrine signaling. Nat Neurosci 22(11):1771-1781PubMed: 31636449 MGI: J:281604

Additional - Gt(ROSA)26Sor^{tm1(CAG-CHRM4,-mCitrine)Ute}* related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Probe:Gt(ROSA)26Sor Probe
- Standard PCR:Gt(ROSA)26Sor
- Genotyping resources and troubleshooting
Dietary Information
- LabDiet® 5K52 formulation (6% fat)
Breeding Considerations

When maintaining a live colony, heterozygous mice may be bred together or to wildtype mice from the colony, or to C57BL/6NJ inbred animals (Stock No. 005304). While homozygous mice are expected to be viable and fertile, the generation of homozygous mice has not been attempted to date (January 2015).
- Additional Breeding and Husbandry Support
Mating System
- Wild-type x Heterozygote
- Heterozygote x Wild-type
Citation
When using the R26-LSL-Gi-DREADD , R26-LSL-hM4Di-DREADD mouse strain in a publication, please cite the originating article(s) and include JAX stock #026219 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

AX12 (Maximum)

Pricing & Availability

Available

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International

Live Mouse

Age

Genotype

Price

weeks

Breeder Pair

Sex

Genotype

Price

Female
Male

Cryorecovery - Pricing

Service/Product	Description	Price
	Heterozygous or wildtype for Gt(ROSA)26Sor<tm1(CAG-CHRM4*,-mCitrine)Ute>

Related Products and Services

Frozen Mouse Embryo	B6N.129-Gt(ROSA)26Sor<tm1(CAG-CHRM4*-mCitrine)Ute>/J	$2595.00
Frozen Mouse Embryo	B6N.129-Gt(ROSA)26Sor<tm1(CAG-CHRM4*-mCitrine)Ute>/J	$2595.00
Frozen Mouse Embryo	B6N.129-Gt(ROSA)26Sor<tm1(CAG-CHRM4*-mCitrine)Ute>/J	$3373.50
Frozen Mouse Embryo	B6N.129-Gt(ROSA)26Sor<tm1(CAG-CHRM4*-mCitrine)Ute>/J	$3373.50

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Also Known As:R26-LSL-Gi-DREADD , R26-LSL-hM4Di-DREADD, R26-hM4Di/mCitrine

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Additional Information

Selected References

Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute

Allele Symbol: Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

Genotype: Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute/Gt(ROSA)26Sor+B6N.129-Gt(ROSA)26Sor/J

normal phenotype

References

Additional References

Additional - Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute related

Genotyping Protocols

Dietary Information

Breeding Considerations

Mating System

Citation

Animal Health Reports

Live Mouse

Breeder Pair

Cryorecovery - Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms Of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability

Gt(ROSA)26Sor^{tm1(CAG-CHRM4,-mCitrine)Ute}*

Allele Symbol: Gt(ROSA)26Sor^{tm1(CAG-CHRM4,-mCitrine)Ute}*

Genotype: Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute}/Gt(ROSA)26Sor⁺
B6N.129-Gt(ROSA)26Sor/J

Additional - Gt(ROSA)26Sor^{tm1(CAG-CHRM4,-mCitrine)Ute}* related