Multicolor Luciferase Assay System: One-Step Monitoring of Multiple Gene Expressions with a Single Substrate

Abstract

Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control. We have developed a novel reporter assay system in which three luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.

In Vivo Cell Tracking with Bioluminescence Imaging

“…BLI has been further applied to the in vivo study for simultaneous monitoring of multiple cellular events. Nakajima et al [56] developed a novel reporter assay system that emits three different colors (green, orange, and red light) using a single substrate. They demonstrated that this system enabled direct comparison of two or more transcriptional activities and/or interactions with transcription factors in the same cell population in a one-step reaction with a single luciferin.…”

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

“…10 The bioluminescent spectra of WT and mutant luciferases from transfected NIH3T3 cell extracts were evaluated. All mutants showed the similar spectral shape as WT; three (I212L, S463R, and I212L/S463R) had the same emission maximum as WT (kmax ca.…”

“…10,11 The optimized cDNA in mammalian expression vector (pCMV-SLR), whose expression is driven by the CMV promoter, was used as the template for mutation. Site-directed mutagenesis was performed using a KOD-Plus-Mutagenesis Kit (Toyobo) according to the manufacturer's instructions.…”

“…7 They are also an important component for constructing multicolor reporter systems, which are vital for monitoring multiple genes. [8][9][10][11][12][13] Beetle luciferases are intriguing because, in the presence of ATP and Mg þþ , they can efficiently catalyze D-luciferin oxidation emitting visible light from green to red. 14 Luciferase cDNAs have been cloned from many beetles, but only the one from the railroad worm Phrixothrix hirtus can produce naturally red-emitting luciferase (PhRED) and has an additional advantage of spectral pH insensitivity.…”

“…10,11 Nevertheless, the poor thermostability of wild-type (WT) PhRED, which is also a common problem for all other beetle luciferases 23 and can affect the detection efficiency of in vivo BLI, 29 limits its application in demanding circumstances. Some attempts have been made to improve firefly luciferase thermostability by mutagenesis (e.g., Ppy, [30][31][32] Lcr, 33 Luciola lateralis (Lla), 34 Hotaria parvula (Hpa) 35 ).…”