A Split sfGFP-Based Signal-Amplified Reporter System for Enhanced Detection of Promoter Activity

Fluorescent reporter systems for protein expression enable real-time, dynamic, and single-cell visualization of gene expression in living cells, but lack sufficient sensitivity for detecting low-abundance proteins. Herein, we developed a signal-amplified split superfolder green fluorescent protein (sfGFP) reporter system: sfGFP was split into the GFP_1-10 fragment driven by a constitutively strong promoter and a 20× tandem GFP₁₁ tag controlled by weak promoters, with the incorporation of coiled-coil dimerization domains to facilitate fragment complementation. This design enhanced fluorescent signals by approximately 10-fold, significantly improving detection sensitivity and enabling clear visualization of green fluorescence even at extremely low mRNA levels. Application of this system to evaluate Tet-On inducible systems revealed substantial basal leakage in Tet-On 2G. Moreover, real-time monitoring of endogenous weakly promoters (e.g., VIM, HPRT1, GUSB) demonstrated that the fluorescent intensity was approximately 10-fold higher than that of conventional reporter systems. This platform provides a highly sensitive and versatile tool for weak promoter activity analysis, dynamic tracking of low-abundance genes, and investigation of cell types and states.