Abstract
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.