Scientists measured ribosomal out-of-frame protein synthesis in 1-cell stage zebrafish embryos using IVT mRNA expressing in-frame firefly luciferase (NFluc) and a catalytically inactive out-of-frame luciferase (CFluc). These mRNAs translate as efficiently as unmodified mRNAs.
What is FLuc?
FLuc stands for firefly luciferase, and it refers to an enzyme that emits light when provided with the substrate luciferin and oxygen. Firefly luciferase is commonly used as a reporter gene in molecular biology and biotechnology applications, particularly in gene expression and promoter activity studies.
FLuc is often employed in bioluminescence assays, where a promoter of interest controls the expression of the FLuc gene. The light emitted by FLuc can then be quantified, measuring gene expression.
How is FLuc Decoded?
The FLuc mRNA sequence encodes for firefly luciferase, which is expressed in cells after transfection and catalyzes the oxidation of luciferin to produce oxyluciferin, generating yellow-green bioluminescence. The fluorescence intensity of the oxyluciferin is proportional to the luciferase expression level. Thus, FLuc mRNA can assess cellular translation in cell culture, flow cytometry, and microscopy experiments. It can also be used as a positive control to analyze the stability and sensitivity of mRNA delivery formulations or expression systems.
The luciferase activity of the mRNA can be measured in the cell culture or flow cytometry experiments by determining the number of GFP-positive (EGFP+) cells. The fluorescence intensity of the EGFP is proportional to the luciferase activity of the cells and can be used to determine the amount of mRNA synthesized in the cell. Moreover, the mRNA can be fused with a green fluorescent protein (GFP) gene to express the luciferase and the GFP in the same cell.
The cryptic promoter activity of the FLuc gene can also be detected by Northern blotting or real-time qRT-PCR.
The FLuc gene is fused with a gene’s promoter region under investigation in a typical reporter gene system. When the promoter is active, FLuc is transcribed and translated, leading to the production of the enzyme. The addition of luciferin and oxygen allows FLuc to catalyze a reaction that produces light.
What is the FLuc mRNA Sequence?
The FLuc mRNA sequence encodes for firefly luciferase, a protein that is excited by light and produces bioluminescence. It is commonly used in cell biology studies to quantify gene expression and monitor cell viability. This mRNA is capped with a poly(A) tail to improve its stability and translation efficiency in mammalian cells.
Northern blot analysis of poly(A)+-enriched RNA isolated from CCL13 cells transfected with pFG, pFG(-P), pRGLuc, or their promoterless variants reveals that transcripts complementary to the FLuc CDS are produced in a step-wise manner. This result is due to cryptic transcription initiated from several cryptic transcription initiation sites within the coding region of the FLuc gene.
Another way to detect FLuc mRNA transcripts is by using fluorescence microscopy or real-time qRT-PCR. The red cells indicate that the mRNA was successfully expressed and translated into the FLuc protein, producing a high level of bioluminescence.
Alternatively, researchers can use FLuc mRNA to assess protein synthesis in vivo by injecting it into genetically modified Ai14 mice. This model allows researchers to screen for mRNA expression in tissues with minimal invasiveness and can be used for longitudinal studies to monitor mRNA expression over time.
How to Decode the FLuc mRNA Sequence
The firefly luciferase (FLuc) gene from the common firefly Photinus pyralis has become one of the most commonly used reporter genes in thousands of different experiments worldwide due to its sensitivity, versatility, and relative simplicity of use. As an alternative to expensive, high-throughput chromosomal transfection, many commercially available FLuc-based reagents have been introduced.
However, analyzing samples with Northern or Western blots shows diffuse signals rather than the expected distinct bands, suggesting that the 1653-bp FLuc+ coding sequence contains several cryptic transcription sites. This observation is confirmed by qRT-PCR, demonstrating no strictly localized transcription site but uniformly distributed amplicons across the coding region.
To determine whether these cryptic transcripts can be translated into enzymatically active polypeptides, researchers performed translation experiments with unmodified and 1-methylPs Fluc+1FS2 mRNAs containing either the U*187C or U*208C slippery site mutation. Both mutant mRNAs exhibit +1 frameshifting activity comparable to the WT Fluc mRNA. In contrast, the translation of mRNAs with the U*187C or U*208C double-mutant slippery site yields no detectable +1 frameshifting and no significant change in the amino acid sequence of the predicted product.
The results from these experiments clearly show that the FLuc cryptic promoter cannot be efficiently translated into a functional polypeptide. In contrast, expression of the full-length EGFP gene controlled by an intact CMV IE promoter leads to robust green fluorescent cell (GFP) production. The data also indicate that the cryptic promoter is only weakly expressed in liver cells.
It’s important to note that using FLuc as a reporter gene is just one of many molecular biology and biotechnology techniques. Researchers often choose reporter genes based on the specific requirements of their experiments and the detection methods available.