Novel floxed cannabinoid receptor 2 mouse line combines knockout capability with dual fluorescent reporters

Background The cannabinoid receptor 2 (CB 2 ) is involved in regulating immune responses, yet its specific function in microglia remains poorly defined. This study aimed to generate and validate a microglia-specific, inducible CB 2 knockout mouse model incorporating reporter genes to enable precise detection of CB 2 expression and CB 2 knockout. Methods A novel floxed CB 2 mouse line was generated, incorporating GFP and tdTomato reporter genes driven by the Cnr2 promoter to indicate CB 2 expression and CB 2 knockout, respectively. This line was crossed with Cx3cr1 or Tmem119 tamoxifen-inducible Cre lines to achieve macrophage- or microglia-specific CB 2 knockout, respectively. Behavioural testing, in vitro assays, sequencing and in vivo immunofluorescence were used to assess the efficiency and specificity of CB 2 knockout as well as potential off-target effects. Results The floxed allele did not alter breeding or motor behaviour in mice, nor CB 2 function. CB 2 expression, indicated by GFP, followed expected patterns across tissues and conditions. Sequencing revealed both DNA and RNA of the floxed allele was as anticipated. Tamoxifen-induced Cre activity successfully initiated tdTomato expression exclusively in microglia of tamoxifen treated, Cre positive mice, validating the specificity and inducibility of CB 2 knockout. Microglial tdTomato expression confirmed successful CB 2 knockout in 9.3% of TmemCB 2 and 91.7% of Cx3CB 2 microglia. Peripheral tdTomato expression persisted beyond 3 weeks post-tamoxifen in Cx3CB 2 mice but was minimal in TmemCB 2 mice. Conclusion This novel microglia-specific, inducible CB 2 knockout model is the first to combine a floxed CB 2 allele with reporter genes, an essential advancement given the lack of reliable CB 2 antibodies. The findings demonstrate the model’s specificity and effectiveness, while highlighting important considerations regarding Cre-mediated effects and recombination specificity. Furthermore, the floxed mouse can be crossed with any Cre line to study CB 2 expression and function in various tissues. This model provides a powerful platform for advancing understanding of CB 2 roles in microglia and supports future exploration of CB 2 -targeted therapeutic strategies.