STOCK P2ry1^{tm1.1(cre)Lbrl}/RcngJ

Stock number: 029284
Product name: P2ry1-ires-Cre
Research areas: Internal/Organ Research, Neurobiology Research, Research Tools

Description

P2ry1-ires-Cre knock-in mice express Cre recombinase under the direction of the P2ry1 promoter in vagal sensory neurons in the lung and brain stem that control respiration.

Detailed description

P2ry1-ires-Cre mice have an IRES-Cre fusion protein inserted 3 base pairs downstream of the stop codon of the purinergic receptor P2Y, G-protein coupled 1 (P2ry1) gene. The presence of IRES-Cre cassette downstream of the stop codon allows for expression of P2RY1 and cre. P2RY1 encodes a receptor present on many cell types, including a subset of fast-conducting myelinated A fiber vagal sensory neurons in the lung and brain stem. The P2ry1-ires-Cre reporter is expressed in 94% of P2ry1 neurons when bred to a cre-dependent EYFP reporter. 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 P2ry1-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 P2RY1 neurons silences respiration for 7.9 seconds, trapping animals in exhalation without impacting heart rate or gastric pressure, which are also under vagal control. Activation causes a 72% decrease in respiration rate, a 67% decrease in minute volume, and a 5.5% increase on tidal volume.

 

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 purinergic receptor P2Y, G-protein coupled 1 (P2ry1) 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 P2ry1-ires-Cre mice were bred to C57BL/6J mice for at least 1 generation. 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: P2ry1^{tm1.1(cre)Lbrl}
Name: targeted mutation 1.1, Stephen Liberles
MGI accession ID: MGI:5776536
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: P2ry1
Marker name: purinergic receptor P2Y, G-protein coupled 1
Chromosome: 3
Strain of origin: 129S6/SvEvTac
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre recombinase is expressed in vagal sensory neurons in the lung and brain stem.
Molecular note: A cassette encoding IRES, Cre recombinase, and an FRT-flanked neomycin selection cassette is inserted into the mouse P2ry1 locus 3 base pairs downstream of the coding sequence stop codon.