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High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Published on: June 1, 2014
Firefly luciferase gene contains a cryptic promoter
Václav Vopálenský1, Tomás Masek, Ondrej Horváth
1Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.
RNA (New York, N.Y.)
|August 14, 2008
Summary
A cryptic promoter activity was discovered within the firefly luciferase (FLuc) gene, impacting RNA-based research. Researchers should exercise caution when designing FLuc assays due to this finding.
Area of Science:
- Molecular Biology
- Cell Biology
- Reporter Gene Assays
Background:
- Firefly luciferase (FLuc) is a widely used reporter in molecular and cellular biology.
- The luc+ gene is the most common version of FLuc used in research.
Purpose of the Study:
- To report and characterize cryptic promoter activity within the FLuc gene.
- To discuss the implications of this cryptic promoter on experimental results, particularly in RNA-focused research.
Main Methods:
- Investigated cryptic promoter activity of the FLuc gene in mammalian (human CCL13, Huh7) and yeast cells.
- Quantified cryptic transcription levels relative to the cytomegalovirus (CMV) promoter.
Main Results:
- The FLuc coding region exhibits cryptic promoter activity in both mammalian and yeast cells.
- Cryptic transcription from the luc+ gene was found to be 10-16 times weaker than the strong immediate-early CMV promoter in human cells.
- This activity may influence studies on translation initiation and miRNA/siRNA function.
Conclusions:
- The newly identified cryptic promoter in the FLuc gene necessitates careful assay design in firefly luciferase-based experiments.
- This finding may offer new possibilities for experiments involving the hepatitis C virus internal ribosome entry site (HCV IRES).
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