Firefly luciferase gene contains a cryptic promoter

Abstract

A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10-16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferasebased assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now.

The Luc2 gene enhances reliability of bicistronic assays

“…All of the methods and materials used in this study were described previously [3]. Here, we briefly summarize these protocols with emphasis on the differences between this and the previous study.…”

“…We reported, and characterized in detail, a significant cryptic promoter activity in the luc+ coding region that is detectable in mammalian as well as yeast cells [3]. More importantly, cryptic transcription from the luc+ gene was only ten to sixteen times weaker than the strong, immediate-early cytomegalovirus promoter expression in human CCL13 and Huh7 cells.…”

“…The pRG plasmid and its promoter-less variant pRG(-P) are bicistronic vectors containing the DsRED2 and EGFP genes (Clontech) as the first and the second cistrons, respectively [3,21]. The complete vector sequences are deposited under the IRESite IDs 368 and 369 (www.IRESite.org).…”

“…A promoter-less variant of the latter construct, pRG(-P)Luc2, was created by removing the CMV IE promoter by VspI/NheI double-digestion, vector blunting and re-ligation. The pRGLuc+ and pRG(-P)Luc+ vectors were described previously [3]. All of the vectors were verified by restriction endonuclease digestion and sequencing.…”

“…Details of the cultivation are described in previous work [3]. For the flow cytometry analyses, cells were plated in 24-well tissue culture plates 24 hours before transfection.…”