C57BL/6N-Tg(Ins2-Chrm3*)QJwe/J

Stock number: 028964
Product name: β-rhM3Dq Tg (or β-R-q Tg)
Research areas: Cancer Research, Cell Biology Research, Diabetes and Obesity Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

These β-rhM3Dq Tg (or β-R-q Tg) mice express the G protein-coupled receptor DREADD rhM3Dq (also called R-q) directed to pancreatic β-cells by the rat insulin 2 promoter. rhM3Dq induces the canonical G_q/11 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.

Detailed description

The β-rhM3Dq (or β-R-q) transgene has the rat insulin 2 promoter directing expression of a hemagglutinin epitope-tagged, mutant G protein-coupled receptor (rhM3Dq or R-q) to pancreatic β-cells. The rhM3Dq is a G_q-coupled rat/human chimer muscarinic 3 DREADD harboring two amino acid substitutions (Y149C/A239G) 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 rhM3Dq induces the selective activation of the canonical G_q/11 signaling pathway, leading to increased intracellular Ca+ and IP levels.

Mice that are hemizygous for the transgene (β-rhM3Dq Tg or β-R-q 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-rhM3Dq Tg with a similarly created transgenic mouse that expresses a rat insulin 2 promoter-driven rM3Ds (β-GsD Tg ; Stock No. 028966).

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 G_q/11 or Gα_s signaling pathways, respectively, in a conditional and β-cell-selective fashion. Specifically, following CNO administration in β-rhM3Dq Tg, the canonical G_q/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 Gα_s pathway in GsD-expressing pancreatic β-cells results in qualitatively similar in vivo metabolic effects.

Development

The β-R-q transgene was designed in the laboratory of Dr. Jürgen Wess (NIH-NIDDK) to have (from 5' to 3') a 650 bp rat insulin 2 (Ins2; RIP II) promoter fragment, a hemagglutinin epitope tag (HA), the rhM3Dq coding sequence (R-q; described below), and untranslated human growth hormone sequences (containing transcriptional termination, polyadenylation and splicing signals).

The 4.6 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 Q). The β-rhM3Dq Tg (or β-R-q Tg) colony was backcrossed onto C57BL/6NTac for seven generations, and then frozen as sperm. In 2016, β-rhM3Dq Tg sperm was sent to The Jackson Laboratory Repository. Upon arrival, our β-rhM3Dq 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 rhM3Dq (or R-q) sequence is a G_q-coupled rat/human chimer M3 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the rhM3Dq sequence, a 924 bp sequence of the wildtype rat muscarinic 3 receptor (Chrm3; M3R) was replaced with the human M3R sequences containing the Y149C and A239G amino acid substitutions (corresponding to rat Y148C and A238G). These mutations 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).

Allele

Symbol: Tg(Ins2-Chrm3*)QJwe
Name: transgene insertion Q, Jurgen Wess
MGI accession ID: MGI:5766541
Generation method: Transgenic
Attributes: Inserted expressed sequence
Marker symbol: Tg(Ins2-Chrm3*)QJwe
Marker name: transgene insertion Q, Jurgen Wess
Chromosome: UN
Strain of origin: C57BL/6NTac
Expressed genes: Chrm3,cholinergic receptor, muscarinic 3,rat; HA tag,HA tag,
Site of expression: Pancreatic beta cells.
Molecular note: This 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 rhM3Dq coding sequence (R-q; described below), and untranslated human growth hormone sequences (containing transcriptional termination, polyadenylation and splicing signals). The transgene is expressed in pancreatic beta cells.The rhM3Dq (or R-q) sequence is a Gq-coupled rat/human chimer M3 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the rhM3Dq sequence, a 924 bp sequence of the wildtype rat muscarinic 3 receptor (Chrm3; M3R) was replaced with the human M3R sequences containing the Y149C and A239G amino acid substitutions (corresponding to rat Y148C and A238G). These mutations 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).