B6N;129-Tg(CAG-CHRM3*,-mCitrine)1Ute/J

Stock number: 026220
Product name: CAG-LSL-Gq-DREADD , R26-LSL-Gq-DREADD , R26-LSL-hM3Dq-DREADD
Strain synonyms: R26-hM3Dq/mCitrine
Research areas: Cancer Research, Cell Biology Research, Diabetes and Obesity Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

The CAG-LSL-Gq-DREADD allele (formerly called R26-LSL-Gq-DREADD) allows Cre recombinase-inducible expression of a CAG promoter-driven HA-hM3Dq-pta-mCitrine. Following cre-mediated removal of an upstream floxed-STOP cassette, expression of HA-tagged hM3Dq and mCitrine yellow fluorescent protein is observed. hM3Dq is a mutant G protein-coupled receptor (GPCR) that induces the canonical G_q pathway specifically following administration of the pharmacologically inert molecule clozapine-N-oxide (CNO). These CAG-LSL-Gq-DREADD mice may be useful for chemogenetic/pharmacogenetic studies to express an activating DREADD that effectively induces firing of neurons.

Note on allele, transgene insertion and copy number: This 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. Furthermore, the transgene integrated as a single (or partial) copy on chromosome 14 [~41 Mb] and duplicated nearby genes - see Detailed Description section for details.

Detailed description

Note on allele, transgene insertion and copy number: 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. Furthermore, The Jackson Laboratory colony was screened to determine the transgene integration site(s). This analysis revealed a single (or partial) transgene copy integrated on chromosome 14 (chr14:41,019,438-41,024,312 [mm10]) in a manner suggestive of complex structural variation surrounding the integration site. Specifically, there is duplication of nearby genome sequences involving the genes Tspan14, Prxl2a, Dydc2, Dydc1, Mat1a, Sftpa1 and Mbl1.

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.

HA Expression Before and After Cre ; SOP for Brain Immunofluorescence: In 2022, The Jackson Laboratory bred some CAG-LSL-Gq-DREADD hemizygous females with Sox2-Cre hemizygous males (Stock No. 008454). The resulting offspring were used to examine HA-hM3Dq expression via HA antibody immunofluorescent imaging. The Cre-negative CAG-LSL-Gq-DREADD hemizygous offspring had weak occasional HA staining, while the Cre-positive CAG-LSL-Gq-DREADD hemizygous offspring showed positive HA staining throughout brain that was much brighter than Cre-negative littermates and much brighter than the 026220 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: 026220 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].

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).

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).

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

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

Development

Note on allele, transgene insertion and copy number: 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. Furthermore, The Jackson Laboratory colony was screened to determine the transgene integration site(s). This analysis revealed a single (or partial) transgene copy integrated on chromosome 14 (chr14:41,019,438-41,024,312 [mm10]) in a manner suggestive of complex structural variation surrounding the integration site. Specifically, there is duplication of nearby genome sequences involving the genes Tspan14, Prxl2a, Dydc2, Dydc1, Mat1a, Sftpa1 and Mbl1.

The CAG-LSL-Gq-DREADD mice (formerly called R26-LSL-Gq-DREADD) were designed by Dr. Ute Hochgeschwender (while at Duke University). The Rosa-CAG-LSL-HA-hM3Dq-pta-mCitrine 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 hM3Dq sequence (see strain description for additional information), a porcine teschovirus (PTA) cleavage site, the mCitrine fluorescent protein (see strain description for additional information), 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 construct was originally designed to be 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 (see important note). ES cells were injected into recipient blastocysts, and the resulting chimeric animals were bred with C57BL/6NCrl mice. The donating investigator reported that CAG-LSL-Gq-DREADD mice were further crossed to C57BL/6NCrl for several generations prior to sending CAG-LSL-Gq-DREADD males from generation N4 to The Jackson Laboratory Repository in 2015 (see SNP results below). 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 CAG-LSL-Gq-DREADD mice were then bred together, to wildtype mice from the colony or to C57BL/6NJ inbred animals. The donating investigator reports the Y chromosome may not yet be from the C57BL/6N background.

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/6N and C57BL/6J substrains, was performed on the first generation rederived living colony at The Jackson Laboratory Repository. This revealed some mice had 3 of 27 markers that were not fixed for C57BL/6 allele-type: 2 markers were segregating for 129 allele-type (either 129X1 or 129S1) on chromosomes 2 [~27.3 Mbp] and 12 [~9.8 Mbp], and one marker on chromosome 19 [~13.1 Mbp] was segregating for 129X1 allele-type. The 5 markers that determine C57BL/6N from C57BL/6J were all found to be C57BL/6N allele-type. These data suggest that the mice sent to The Jackson Laboratory Repository were not completely backcrossed onto the C57BL/6N genetic background, and also that the targeted mutation may have integrated onto the 129X1-derived homologous chromosome when electroporated into the (129X1/SvJ x 129S1/Sv)F1 ES cells.

Allele

Symbol: Tg(CAG-CHRM3*,-mCitrine)1Ute
Name: transgene insertion 1, Ute Hochgeschwender
MGI accession ID: MGI:5614052
Generation method: Transgenic
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Tg(CAG-CHRM3*,-mCitrine)1Ute
Marker name: transgene insertion 1, Ute Hochgeschwender
Chromosome: 14
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: YFP,Yellow Fluorescent Protein,jellyfish; CHRM3,cholinergic receptor muscarinic 3,human
Site of expression: Yellow fluorescent protein variant mCitrine and the hemagglutinin epitope-tagged, mutant G protein-coupled receptor hM3Dq (modified CHRM3 sequence) will be expressed in tissues expressing Cre recombinase.
Molecular note: The construct 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 hM3Dq 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.

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).

Citrine 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).

Aanalysis of the transgene integration site at The Jackson Laboratory revealed a single (or partial) transgene copy integrated on chromosome 14 (chr14:41,019,438-41,024,312 [mm10]) in a manner suggestive of complex structural variation surrounding the integration site. Specifically, there is duplication of nearby genome sequences involving the genes Tspan14, Prxl2a, Dydc2, Dydc1, Mat1a, Sftpa1 and Mbl1