Development of a Cre-recombination-based color-switching reporter system for cell fusion detection.

ABSTRACT

Cell fusion plays a key role in the development and formation of tissues and organs in several organisms. Skeletal myogenesis is assessed in vitro by cell shape and gene and protein expression using immunofluorescence and immunoblotting assays. However, these conventional methods are complex and do not allow for easy time-course observation in living cells. Therefore, this study aimed to develop a Cre recombination-based fluorescent reporter system to monitor cell-cell fusion. We combined green and red fluorescent proteins with a Cre-loxP system to detect syncytium formation using a fluorescent binary switch. This allowed us to visualize mononucleated cells with green fluorescence before fusion and multinucleated syncytia with red fluorescence by conditional expression after cell fusion. The formation of multinuclear myotubes during myogenic differentiation was detected by the change in fluorescence from green to red after Cre-mediated recombination. The distribution of the fluorescence signal correlated with the expression of myogenic differentiation markers. Moreover, red reporter fluorescence intensity was correlated with the number of nuclei contained in the red fluorescent-positive myotubes. We also successfully demonstrated that our fusion monitoring system is applicable to the formation of skeletal muscle myotube and placental syncytiotrophoblast. These results suggest that the color-switching fluorescent reporter system, using Cre-mediated recombination, could be a robust tool used to facilitate the study of cell-to-cell fusion.