Combining intracellular and secreted bioluminescent reporter proteins for multicolor cell-based assays

Bioluminescent (BL) proteins are a promising tool for diverse applications based on reporter gene technology thanks to their high sensitivity and range of linear response. Due to their widespread use in the environmental, medical and agro-food fields, there is a great need for new BL reporter proteins with improved characteristics to provide researches a wide range of suitable reporters. Few efforts have been made in this direction and further improvement of BL reporter features (e.g., thermostability, narrower emission bandwidth, emission at different wavelengths) tailored for specific applications would be a remarkable progress toward the development of ultrasensitive multiplexed assays either in vitro or in vivo. The suitability of using red- and green-emitting thermostable mutants of Photinus pyralis firefly luciferase and two click beetle luciferases in combination with a secreted luciferase from Gaussia princeps was evaluated to develop a triple-color mammalian assay. Two triple-reporter model mammalian systems were developed in a human hepatoblastoma cell line to monitor the transcriptional regulation of cholesterol 7-alpha hydroxylase (cyp7a1), the enzyme that catalyzes the first and rate-limiting step of the main pathway responsible for cholesterol degradation in humans. These model systems allowed us to evaluate the feasibility of using two intracellular BL reporters and a secreted one in the same cell-based assay. The selection of reporter proteins characterized by similar expression levels was identified as a critical point for the development of a multicolor assay.