Chemokines are a class of small, secreted cytokines that regulate cell migration and positioning, thereby playing central roles in development and immune homeostasis. In addition, they interact with various extracellular factors and can modulate neural activity during inflammation. Among them, C-X-C motif chemokine ligands 9, 10, and 11 (CXCL9-11) bind to the C-X-C motif chemokine receptor 3 (CXCR3), a member of the G protein-coupled receptor (GPCR) superfamily, mediating diverse functions including T cell homing, proliferation, and angiogenesis. GRAB-LoX3-1.0 (GPCR activation-based sensor for Ligands of CXCR3; also called LoX3-1.0 or LSL-LoX3-1.0) reporter mice were engineered to have Cre-inducible expression of a genetically encoded fluorescent sensor for endogenous CXCR3 ligands. This sensor, developed based on the GRAB (GPCR activation-based) design principle, consists of a circularly permuted green fluorescent protein (cpEGFP) inserted into the third intracellular loop of CXCR3, enabling the ligand-induced conformational change to be converted into a measurable optical signal. An mCherry protein is fused to the N-terminus of LoX3-1.0 via a flexible (GG) linker to normalize expression levels and label the plasma membrane, enabling ratiometric fluorescence measurement. Following Cre recombinase-mediated excision of a loxP-flanked transcriptional stop cassette, the GRAB-LoX3-1.0 sensor exhibits a high signal-to-noise ratio in fluorescence response and nanomolar affinity for CXCR3 ligands. The sensor offers temporal resolution on the order of tens of milliseconds and submicron spatial resolution. LoX3-1.0 can respond to all three natural CXCR3 ligands including CXCL9, 10 and 11, and also shows similar activation potency as CXCR3 in responding to truncated variants of CXCL10.

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GRAB-LoX3-1.0 (also called LoX3-1.0 or LSL-LoX3-1.0) reporter mice express a genetically-encoded GPCR activation-based (GRAB) fluorescent sensor for CXCR3 ligands upon Cre recombinase-mediated excision of a loxP-flanked transcription stop sequence (LSL). This reporter strain enables the precise examination of chemokine dynamics and spatiotemporal properties in various physiological and pathological conditions.

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