Spectral-Resolved Gene Technology for Multiplexed Bioluminescence and High-Content Screening

Abstract

The availability of new bioluminescent proteins, obtained by cDNA cloning and mutagenesis of wild-type genes, expanded the applicability of these reporters from the perspective of using more proteins emitting at different wavelengths in the same cell-based assay. By spectrally resolving the light emitted by different reporter proteins, it is in fact possible to simultaneously monitor multiple targets. A new luciferase isolated from Luciola italica has been recently cloned, and thermostable red- and green-emitting mutants were obtained by random and site-directed mutagenesis. Different combinations of luciferases were used in vitro as purified proteins and expressed in bacterial and mammalian cells to test their suitability for multicolor assays. A mammalian triple-color reporter model system was then developed using a green-emitting wild-type Photinus pyralis luciferase, a red thermostable mutant of L. italica luciferase, and a secreted Gaussia princeps luciferase (GLuc) to monitor the two main pathways of bile acid biosynthesis. The two firefly luciferases were used to monitor cholesterol 7-alpha hydroxylase and sterol 27-hydroxylase, while secreted constitutively expressed GLuc was used as an internal vitality control. By treating the cells with chenodeoxycholic acid, it was possible to obtain dose-dependent inhibitions of the two specific signals together with a constant production of GLuc, which allowed for a dynamic evaluation of the metabolic activity of the cells. This is the first triple-color mammalian reporter assay that combines secreted and nonsecreted luciferases requiring different substrates, thus avoiding reciprocal interference between different BL signals. This approach is suitable for high content analysis of gene transcription in living cells to shorten the time for screening assays, increasing throughput and cost-effectiveness.

Secreted blood reporters: Insights and applications

“…Many luciferases which are expressed intracellularly and emit different colored light (green and red-emitting luciferases from click beetle (Caysa et al, 2009; Takeuchi et al, 2010) and the American or Italian firefly (Branchini et al, 2007)) have been cloned. Secreted variants of these lucifrases could be engineered and multiplexed with Gluc for multicolor applications using spectral unmixing (Gammon et al, 2006; Michelini et al, 2008). …”

The New Era of Bioluminescence Resonance Energy Transfer Technology

“…Other BRET studies involving various GPCR hetero-dimerization has suggested that Renilla luciferase and EYFP as energy transfer partners is limited as the emission spectrum from the luciferase tends to obscure the energy transfer output from EYFP [47]. Using the same RLuc-EYFP BRET format, HTS assays were developed for evaluation of the estrogen-like and androgen-like activity from biological and environmental samples [48][49][50]. The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in mammalian cells.…”

Current advanced bioluminescence technology in drug discovery

“…Indeed, Nishide et al performed circadian transcription analysis using FLuc and Vargulla luciferase for cultured tissues from transgenic animals [45], and Michelini et al demonstrated a triple reporter gene assay system that was developed in the high-throughput 96-well micro titer plate format combining two different intracellular FLuc variants as reporters and a secretory luciferase as internal control [46,47].…”