High Sensitivity and Low-Cost Flavin luciferase (FLUX)-based Reporter Gene for Mammalian Cell Expression

Abstract

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most common, and thus most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase for Mammalian Cell Expression (FLUX) by engineering luciferase from Vibrio campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells. We found that the FLUX reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUX/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We demonstrated that FLUX can be used for testing inhibitors of the NF-kappa-B signaling pathway, validating FLUX applications for various assays in the future.

Autonomous multicolor bioluminescence imaging in bacteria, mammalian, and plant hosts

“…3 C and 3 D ). Unlike previous reporter assays utilizing Lux (28), which relied on Fluc or Rluc to normalize luminescence intensity and required cell lysis in downstream processes, our NLX system eliminates the need for additional luciferases and the requirement for cell lysis. This feature enhances convenience and cost-effectiveness, making it particularly advantageous for HTS applications.…”