Genome editing-based refinement of GPCR visualization in mice using the oxytocin receptor as a model

Area of Science:

• Molecular Biology
• Neuroscience
• Biophysics

Background:

• G protein-coupled receptors (GPCRs) are crucial for physiological processes and drug development.
• In vivo visualization of GPCRs is hindered by poor antigenicity, limiting research.
• Existing epitope-tagging methods require refinement for effective GPCR tracking.

Purpose of the Study:

• To systematically improve epitope-tagging strategies for in vivo GPCR visualization in mouse models.
• To compare the efficacy of conventional and novel high-sensitivity tags for GPCRs.
• To generate and evaluate genome-edited mouse models for enhanced GPCR imaging.

Main Methods:

• Integration of structural prediction (AlphaFold 3), functional assays, and genome editing.
• Comparison of triple-HA, Spaghetti Monster fluorescent protein (smFP), and ALFA tags fused to the oxytocin receptor (Oxtr).
• Generation of knock-in mice for in vivo assessment of tag performance in tissue staining and neuronal imaging.

Main Results:

• All tested Oxtr variants (triple-HA, smFP, ALFA) retained approximately 70% of wild-type activity in vitro.
• smFP tag significantly improved signal-to-noise ratio in tissue staining compared to conventional tags.
• ALFA tag demonstrated impaired Oxtr trafficking without sensitivity enhancement.
• smFP knock-in mice enabled subcellular visualization of Oxtr distribution in primary cultured neurons.

Conclusions:

• The smFP tag offers a superior strategy for in vivo GPCR visualization, particularly in neuronal contexts.
• Refined genome-edited mouse models enhance the study of GPCR biology.
• This work provides a framework for designing future genome editing approaches for GPCR research.