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 Gi 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.
Ute Hochgeschwender, Central Michigan University
Bryan L Roth, University of North Carolina at Chapel Hill
Genetic Background | Generation |
---|---|
N6+pN1F8
|
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Conditional ready (e.g. floxed), No functional change) | Gt(ROSA)26Sor | gene trap ROSA 26, Philippe Soriano |
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.
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 [Y113C3.33/A203G5.46] 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 Gi 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 Gi 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].
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.
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.
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 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 (Y113C3.33/A203G5.46) 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).
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. |
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). |
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).
Alternatively, homozygous mice are viable and fertile [April 2020] and may be bred together.
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.
Service/Product | Description | Price |
---|---|---|
Heterozygous or wildtype for Gt(ROSA)26Sor<tm1(CAG-CHRM4*,-mCitrine)Ute> |
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 |
Terms are granted by individual review and stated on the customer invoice(s) and account statement. These transactions are payable in U.S. currency within the granted terms. Payment for services, products, shipping containers, and shipping costs that are rendered are expected within the payment terms indicated on the invoice or stated by contract. Invoices and account balances in arrears of stated terms may result in The Jackson Laboratory pursuing collection activities including but not limited to outside agencies and court filings.
The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
What information were you hoping to find through your search?
How easy was it to find what you were looking for?
We may wish to follow up with you. Enter your email if you are happy for us to connect and reachout to you with more questions.
Please Enter a Valid Email Address
Thank you for sharing your feedback! We are working on improving the JAX Mice search. Come back soon for exciting changes.