C57BL/6-Cnr2^tm1.1Geno/J

Stock number: 038343
Product name: CB₂^EGFP/f
Strain synonyms: CB2R^EGFP
Research areas: Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

The CB2^EGFP/f knock-in mice constitutively express an IRES-eGFP fusion sequence under the control of the endogenous cannabinoid receptor 2 (macrophage) gene promoter (Cnr2). These mice also possess loxP sites flanking exon 3 of the Cnr2 gene. These mice may be useful as a fluorescent reporter and in generating conditional mutations for studying neurodegenerative and neuroinflammatory diseases such as Alzheimer's disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS).

Detailed description

Cannabinoid Receptor 2 (CB2), coded by the cannabinoid receptor 2 gene protomer (Cnr2), is a class A G protein-coupled receptor (GPCR) which is expressed primarily in circulating immune cells, the spleen and macrophage-derived cells including osteocytes, osteoclasts, microglia and hepatic Kupffer cells. Endogenous expression is upregulated by proinflammatory and nociceptive stimuli. Increased CB2 receptor expression has been noted in reactive microglia in different neuroinflammatory and neurodegenerative conditions such as neuropathic pain and Alzheimer's disease.

The CB2^EGFP/f knock-in mice express an enhanced green fluorescent protein reporter gene (eGFP) preceded by an internal ribosomal entry site (IRES), that was inserted into the 3’ untranslated region (UTR) of Cnr2. This insertion results in the expression of the eGFP reporter under the control of the endogenous Cnr2 promoter and allows for bicistronic expression of eGFP and Cnr2. In addition, exon 3 and the reporter, are flanked by loxP sites, allowing for conditional inactivation of the gene in cre-expressing tissues. Homozygous CB2^EGFP/f knock-in mice are viable and fertile. The donating laboratory confirmed eGFP expression was detectable by immunofluorescence in specific B-cell populations of the spleen. In the central nervous system, eGFP was very low in homozygous CB2^EGFP/f knock-in mice but is increased when the mice were crossed to a 5xFAD Alzheimer's mouse model (Stock No. 008730). The CB2^EGFP/f/5xFAD progeny demonstrated significant eGFP signal in specific brain regions in concordance with a high-density of beta-amyloid neuritic plaques. These CB2^EGFP/f knock-in mice may be useful for studying the role of CB2 in neurodegenerative and neuroinflammatory diseases.

Development

The CB2^EGFP/f knock-in allele was generated by 3' insertion of an enhanced green fluorescent protein (eGFP) reporter gene into the cannabinoid receptor 2 (macrophage) gene locus (Cnr2). The targeting cassette includes (from 5' to 3') an initial loxP site, 3462 bp-sized fragment containing exon 2 and downstream intronic sequences, a 2980 bp-sized fragment containing the coding part of exon 3 and upstream intronic sequences, and a 3657 bp-sized fragment containing the non-coding part of exon 3 and downstream sequences, followed by an IRES fused to an eGFP sequence, a Frt-flanked neomycin cassette and a second loxP site. The construct was electroporated into C57BL/6-derived embryonic stem (ES) cells. ES cells were injected into C57BL/6J-Tyr^c-2J/J (Stock No. 000058) blastocysts and resulting chimeric males were mated to a C57BL/6-Flp-recombinase mouse line to remove the neomycin cassette. Resulting CB2^EGFP/f mice were maintained on a C57BL/6 genetic background by the donating laboratory (see SNP note below). Upon arrival at The Jackson Laboratory Repository, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

In 2023, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. One of the 5 markers that determine C57BL/6J from C57BL/6N was found to be segregating, on Chromosome 6. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Cnr2^tm1.1Geno
Name: targeted mutation 1.1, Genoway
MGI accession ID: MGI:7424820
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: CB2^EGFP/f; CB2R^eGFP; CB2R-GFP
Marker symbol: Cnr2
Marker name: cannabinoid receptor 2 (macrophage)
Marker synonyms: cannabinoid receptor 2 (spleen); CB2; CB-2; CB2C; CB2-R; CNR2C; CX5; rCB2
Chromosome: 4
Strain of origin: C57BL/6J
Molecular note: An enhanced green fluorescent protein reporter gene (EGFP) preceded by an internal ribosomal entry site (IRES) and followed by an FRT-flanked neomycin gene was inserted into the 3’ untranslated region (UTR) of Cnr2. This results in the expression of the EGFP reporter under the control of the endogenous mouse Cnr2 promoter and transcript from the same bicistronic mRNA as the CB2R protein. In addition, exon 3 and the reporter (including the neomycin) are flanked by loxP sites, allowing for conditional inactivation of the gene in cells expressing cre recombinase. The neomycin gene was removed via flp-mediated recombination.