CMV-CD63-GFP-FLAG Lentivirus

The CMV-CD63-GFP-FLAG lentivirus is a versatile, third-generation self-inactivating lentiviral system designed for robust constitutive expression of the CD63-GFP-FLAG fusion protein driven by the cytomegalovirus (CMV) promoter. This construct offers several key advantages. First, the CMV promoter enables strong and broad transcription in a wide range of human and mammalian cell types, allowing for high-level and uniform expression without the need for induction. Second, lentiviral delivery supports stable genomic integration and long-term expression in both dividing and non-dividing cells, enabling persistent labeling and consistent readouts over time. The fusion construct utilizes CD63, a tetraspanin protein enriched in late endosomes, multivesicular bodies, and extracellular vesicles, to target the GFP reporter protein to exosome-rich compartments for precise visualization of vesicle biogenesis and trafficking. The GFP tag allows for live-cell imaging, rapid expression verification, and quantitative analysis using fluorescence-based methods, while the FLAG epitope provides a compact and well-characterized affinity tag for immunodetection, immunoprecipitation, and gentle capture of labeled vesicles and protein complexes.

This construct is particularly useful for applications requiring the labeling, tracking, and purification of CD63-positive extracellular vesicles (EVs), especially exosomes. In live-cell and time-lapse microscopy, the GFP signal supports visualization of CD63 trafficking, multivesicular body dynamics, and vesicle release at the plasma membrane, enabling quantitative analysis of vesicle biogenesis, cargo loading, and secretion kinetics. In recipient cells, uptake and intracellular trafficking of labeled EVs can be monitored by fluorescence imaging and validated using colocalization with endosomal or lysosomal markers. The FLAG epitope expands the analytical toolkit, allowing for immunocapture and enrichment of CD63-positive vesicles from conditioned media for downstream proteomics, lipidomics, RNA profiling, and functional assays. Researchers can assess and optimize EV isolation methods by comparing total particle counts with GFP-positive vesicle metrics, improving the specificity and recovery of ultracentrifugation, size exclusion, or immunoaffinity chromatography protocols. In cell biology, this system can be used for studying EV pathway perturbations, including screening Rab GTPases, ESCRT components, tetraspanins, and transport regulators, and quickly obtaining results through fluorescence intensity, localization patterns, or vesicle yield.