Dynamic SOX2 gene expression revealed by phase-separated single RNA imaging in live porcine cells

RNA plays a central role in the formation of diverse traits in pigs. Visualizing RNA dynamics at the single-molecule level in living cells is therefore essential for understanding gene-expression regulation in this species. We report an optimized MS2-MCP RNA-labeling system that incorporates a foldon trimerization domain fused to a fluorescent protein and MCP, thereby enhancing fluorescence signals through liquid-liquid phase separation. Using this Foldon-MS2/MCP system, we successfully imaged endogenous GFP mRNA foci in both the nucleus and cytoplasm of living pig cells. Compared with the conventional MS2-MCP system, the Foldon-MS2/MCP system showed markedly increased fluorescence intensity and an improved fluorescence signal-to-noise ratio. We further applied this approach to the endogenous porcine SOX2 gene, achieving high-resolution tracking of SOX2 mRNA foci in live pig cells. Moreover, the system enabled real-time visualization of SOX2 mRNA elongation and translation dynamics. The Foldon-MS2/MCP system provides a powerful platform for investigating RNA localization, gene-expression activation, and endogenous mRNA dynamics in living pig cells.