STOCK Gpr65^{tm1.1(cre)Lbrl}/RcngJ

Stock number: 029282
Product name: Gpr65-ires-Cre
Research areas: Internal/Organ Research, Neurobiology Research, Research Tools

Description

Gpr65-ires-Cre knock-in mice express Cre recombinase under the direction of the Gpr65 promoter in vagal sensory neurons in the gastrointestinal tract that respond to ingested nutrients.

Detailed description

Gpr65-ires-Cre mice have an IRES-Cre fusion protein inserted 3 base pairs downstream of the stop codon of the G-protein coupled receptor 65 (Gpr65) gene. The presence of the IRES-Cre cassette downstream of the stop codon allows for expression of GPR65 and cre. GPR65 encodes a receptor present on several immune cell subsets, and also on a subset of vagal sensory neurons in the gastrointestinal tract that respond to ingested nutrients and control gut constriction and motility. The Gpr65-ires-Cre reporter is expressed in 88% of Gpr65 neurons when bred to a cre-dependent tdTomato reporter (B6;129S6-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J Stock No. 007908) . Homozygous mice are viable and fertile. When these mice are bred with mice containing loxP-flanked sequence, Cre-mediated recombination will result in deletion of the floxed sequences in the Cre-expressing cells of the offspring.

When Gpr65-ires-Cre mice were bred to B6;129S-Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}/J (Stock No. 012569) mice, which express a cre-inducible improved channelrhodopsin-2/EYFP fusion protein, optogenetic activation of GPR65 neurons results in stopped or reduced gastric contractions without impacting breathing or heart rate, which are also under vagal control.

 

Development

A targeting construct was designed by Stephen Liberles (Harvard Medical School) to insert an internal ribosomal entry site (IRES) fused to Cre Recombinase with a polyadenylation signal, followed by a frt-flanked neomycin resistance (neo) cassette, 3 base pairs downstream of the stop codon of the G-protein coupled receptor 65 (Gpr65) gene. This construct was electroporated into 129S6/SvEvTac-derived W4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and the resulting chimeric males were bred to B6;SJL-Tg(ACTFLPe)9205Dym/J mice (Stock No. 003800) to remove the neo cassette. Offspring were crossed to remove the flpe-expressing transgene, and resulting Gpr65-ires-Cre mice were bred to C57BL/6J mice for at least 2 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Gpr65^{tm1.1(cre)Lbrl}
Name: targeted mutation 1.1, Stephen Liberles
MGI accession ID: MGI:5776540
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: Gpr65-ires-Cre
Marker symbol: Gpr65
Marker name: G-protein coupled receptor 65
Marker synonyms: Dig1; Gpcr25; hTDAG8; Tdag8
Chromosome: 12
Strain of origin: 129S6/SvEvTac
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Gpr65 encodes a receptor present on several immune cell subsets, and also on a subset of vagal sensory neurons in the gastrointestinal tract.
Molecular note: A cassette encoding IRES, Cre recombinase, and an FRT-flanked neomycin selection cassette is inserted into the mouse Gpr65 locus 3 base pairs downstream of the coding sequence stop codon.