These β-GsD Tg (or β-R-s Tg) mice express the G protein-coupled receptor DREADD GsD (also called rM3Ds or R-s) directed to pancreatic β-cells by the rat insulin 2 promoter. GsD induces the canonical Gαs signaling pathway specifically following administration of the pharmacologically inert molecule clozapine-N-oxide (CNO). These mice may be useful for chemogenetic/pharmacogenetic applications to express an activating DREADD for studying G protein regulation of β-cell function.
Jürgen Wess, National Institute of Diabetes and Digestive and Kidney Diseases (NIH-NIDDK)
Genetic Background | Generation |
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Allele Type |
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Transgenic (Inserted expressed sequence) |
The β-GsD (or β-R-s) transgene has the rat insulin 2 promoter directing expression of a hemagglutinin epitope-tagged, mutant G protein-coupled receptor (GsD [also called rM3Ds or R-s]) to pancreatic β-cells.
The GsD is a Gs-coupled rat muscarinic 3 DREADD modified to have its second and third intracellular loops replaced with the corresponding turkey β1-adrenergic receptor sequences. Two amino acid substitutions (Y148C and A238G) have been added 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). CNO binding to GsD induces the selective activation of the canonical Gαs signaling pathway, effectively increasing intracellular cAMP levels.
Mice that are hemizygous for the transgene (β-GsD Tg or β-R-s Tg) are viable and fertile. The donating investigator reports they have not tried to generate homozygous mice (May 2016). The phenotype described below compares β-GsD Tg with a similarly created transgenic mouse that expresses a rat insulin 2 promoter-driven rhM3Dq (β-rhM3Dq Tg ; Stock No. 028964).
The transgenic mouse lines β-rhM3Dq Tg (Stock No. 028964) and β-GsD Tg (Stock No. 028966) express similar numbers of rhM3Dq and GsD in their pancreatic islets, respectively. RT-PCR studies confirmed that rhM3Dq and GsD transcripts were not detectable in tissues other than pancreatic islets.
In the absence of CNO, both transgenic lines have normal growth, insulin sensitivity, plasma insulin levels and glucagon levels. While freely-fed β-rhM3Dq Tg have normal blood glucose levels, freely-fed β-GsD Tg have decreased blood glucose levels. Taken together, this indicates the rhM3Dq receptor is devoid of ligand-independent signaling in vivo, whereas the GsD receptor retains some degree of agonist-independent signaling in vivo.
CNO treatment of transgenic mice leads to the activation of β-cell Gq/11 or Gαs signaling pathways, respectively, in a conditional and β-cell-selective fashion.
Specifically, following CNO administration in β-rhM3Dq Tg, the canonical Gq/11 pathway is activated in rhM3Dq-expressing pancreatic β-cells. This leads to significant increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated β-cell mass, associated with pathway-specific alterations in islet gene expression levels. For β-GsD Tg, CNO-induced activation of the canonical Gαs pathway in GsD-expressing pancreatic β-cells results in qualitatively similar in vivo metabolic effects.
The β-R-s transgene was designed in the laboratory of Dr. Jürgen Wess (NIH-NIDDK) to have (from 5prime to 3prime) a 650 bp rat insulin 2 (Ins2; RIP II) promoter fragment, a hemagglutinin epitope tag (HA), the GsD coding sequence (R-s; described below), and untranslated human growth hormone sequences (containing transcriptional termination, polyadenylation and splicing signals).
The 4.2 kbp transgene was microinjected into the pronuclei of ova from fertilized C57BL/6NTac mice. Founder mice were bred with C57BL/6NTac mice, and a germline transmitting founder line was established (default line designation S). The β-GsD Tg (or β-R-s Tg) colony was backcrossed onto C57BL/6NTac for seven generations, and then frozen as sperm. In 2016, β-GsD Tg sperm was sent to The Jackson Laboratory Repository. Upon arrival, our β-GsD Tg transgenic colony may be maintained by breeding hemizygous mice together, to wildtype (noncarrier) mice from the colony, or to C57BL/6NJ inbred mice (Stock No. 005304).
The GsD sequence is a Gαs-coupled rat M3 DREADD (designer receptor exclusively activated by designer drug), also referred to as R-s, rM3Ds, rM3/turkey β1AR chimer DREADD, Gs-DREADD, Gs-D, or D-s. To create the rM3Ds sequence, the wildtype rat muscarinic 3 receptor (Chrm3; M3R) sequence was first modified to replace the second and third intracellular loops with the corresponding turkey β1-adrenergic receptor (ADRB1 or β1AR) sequences. The second modification introduced, via site-directed mutagenesis, the Y148C and A238G amino acid substitutions 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).
Expressed Gene | Chrm3, cholinergic receptor, muscarinic 3, rat |
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Expressed Gene | HA tag, HA tag, |
Site of Expression | Pancreatic Beta-cells. |
Allele Name | transgene insertion S, Jurgen Wess |
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Allele Type | Transgenic (Inserted expressed sequence) |
Allele Synonym(s) | beta-R-s Tg |
Gene Symbol and Name | Tg(Ins2-Chrm3*)SJwe, transgene insertion S, Jurgen Wess |
Gene Synonym(s) | |
Promoter | Ins2, insulin 2, rat |
Expressed Gene | Chrm3, cholinergic receptor, muscarinic 3, rat |
Expressed Gene | HA tag, HA tag, |
Site of Expression | Pancreatic Beta-cells. |
Strain of Origin | C57BL/6NTac |
Chromosome | UN |
Molecular Note | The transgene was designed to have (from 5' to 3') a 650 bp rat insulin 2 (Ins2; RIP II) promoter fragment, a hemagglutinin epitope tag (HA), the GsD coding sequence (R-s; described below), and untranslated human growth hormone sequences (containing transcriptional termination, polyadenylation and splicing signals). The GsD sequence is a Galpha>s<-coupled rat M3 DREADD (designer receptor exclusively activated by designer drug). To create the rM3Ds sequence, the wildtype rat muscarinic 3 receptor (Chrm3; M3R) sequence was first modified to replace the second and third intracellular loops with the corresponding turkey beta1-adrenergic receptor (ADRB1 or beta1AR) sequences. The second modification introduced, via site-directed mutagenesis, the Y148C and A238G amino acid substitutions 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). |
When maintaining a live colony, hemizygous mice may be bred together, to wildtype (noncarrier) mice from the colony, or to C57BL/6NJ inbred mice (Stock No. 005304). To date (May 2016), it has not been attempted to make this strain homozygous.
When using the β-GsD Tg (or β-R-s Tg) mouse strain in a publication, please cite the originating article(s) and include JAX stock #028966 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Hemizygous or non carrier for Tg(Ins2-Chrm3*)SJwe |
Frozen Mouse Embryo | C57BL/6N-Tg(Ins2-Chrm3*)SJwe/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | C57BL/6N-Tg(Ins2-Chrm3*)SJwe/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | C57BL/6N-Tg(Ins2-Chrm3*)SJwe/J Frozen Embryo | $3373.50 |
Frozen Mouse Embryo | C57BL/6N-Tg(Ins2-Chrm3*)SJwe/J Frozen Embryo | $3373.50 |
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