B6(129S6)-P2ry12^{em1(icre/ERT2)Tda}/J

Stock number: 034727
Product name: P2ry12-CreER
Research areas: Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

P2ry12-CreER mice express a tamoxifen-inducible Cre driven by the endogenous purinergic receptor P2Y, G-protein coupled 12 (P2ry12) promoter. These mice are a Cre-lox tool, allowing inducible Cre recombination in P2ry12-expressing cells/tissues (primarily microglia), and may be useful in studying neuroinflammation, neurodegenerative disease, and immune response.

Detailed description

Purinergic receptor P2Y, G-protein coupled 12 (P2ry12) encodes the P2ry12 protein, a microglial-specific GPCR critical for the response of microglia to tissue damage, as well as platelet activation.

The P2ry12-CreER knock-in allele has a tamoxifen-inducible Cre recombinase (CreER^T2) inserted downstream of the P2ry12 translational STOP codon. This is designed to have the endogenous P2ry12 promoter/enhancer elements directing cre/ERT2 expression primarily in microglia, as well as to retain endogenous P2ry12 expression. CreER^T2 fusion gene activity is inducible - observed following tamoxifen administration. As such, when P2ry12-CreER mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences or expression of Cre reporters in P2ry12-expressing cells of the double mutant offspring.

This knock-in allele is designed to avoid disrupting expression of the endogenous protein, though the donating investigator reports preliminary data suggests that the mRNA level for P2RY12 may be slightly reduced in the knock-in mice, while no difference in protein expression is observed between mice heterozygous and homozygous for the P2ry12-CreER allele. Both heterozygotes and homozygotes are viable and fertile with no reported gross physical or behavioral abnormalities.

The donating investigator reports tamoxifen-induced expression primarily in brain parenchymal microglia, with very limited expression in peripheral monocyte-derived or borderzone macrophages, as well as very low expression in other tissues including liver, thymus, and spleen (McKinsey et al, 2020). Thus, these P2ry12-CreER mice may provide a useful alternative to Cx3cr1-Cre strains for the study of resident microglia in the central nervous system.

Development

The P2ry2a-iCreER allele was created in the laboratory of Dr. Thomas D. Arnold, using CRISPR/Cas9 endonuclease-mediated genome editing to target the stop codon of the P2ry12 locus on chromosome 3. The cas9-RNP complex and a 2A-iCreERT2 dsDNA (encoding a viral 2A oligopeptide that mediates ribosomal skipping and the iCreER fusion gene with ~1.5 kbp homology arms) were injected into the pronuclei of C57BL/6N mouse embryos, followed by adoptive embryo transfer. Founder mice and progeny were screened by PCR, and proper genomic integration was verified by Southern blot and genomic sequencing. Founders were bred to C57BL/6J mice for germline transmission. The line was subsequently crossed to a tdTomato reporter on a congenic C57BL/6J background (Ai14; Stock No.007914) and then backcrossed once to C57BL/6J to remove the fluorescent reporter before males were sent to The Jackson Laboratory in 2020. 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: P2ry12^{em1(icre/ERT2)Tda}
Name: endonuclease-mediated mutation 1, Thomas D Arnold
MGI accession ID: MGI:6379582
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible
Synonyms: P2ry12-CreER; P2Y12-CreER
Marker symbol: P2ry12
Marker name: purinergic receptor P2Y, G-protein coupled 12
Marker synonyms: 2900079B22Rik; 4921504D23Rik; ADPG-R; BDPLT8; HORK3; P2T(AC); P2Y(12)R; P2Y(AC); P2Y(ADP); P2Y(cyc); P2y12; SP1999
Chromosome: 3
Strain of origin: C57BL/6N
Molecular note: The allele was created using CRISPR/cas9 endonuclease-mediated genome editing to target the stop codon of the P2ry12 locus on chromosome 3. The cas9-RNP complex and a 2A-iCreERT2 dsDNA (encoding a viral 2A oligopeptide that mediates ribosomal skipping and the iCreER fusion gene with ~1.5 kbp homology arms) were injected into the pronucleus of C57BL/6N mouse embryos, followed by adoptive embryo transfer. Founder mice and progeny were screened by PCR, and proper genomic integration was verified by Southern blot and genomic sequencing. Founders were bred to C57BL/6J mice for germline transmission.