The Significance of eGFP mRNA in Gene Expression Studies

GFP enables the detection of individual cells on a flow cytometer and thus offers unique possibilities for gene expression analysis in single-cell settings. This provides a more complete picture of promoter activity than measurements that only offer mean values for the cell population.

Time-courses of eGFP expression after mRNA or pDNA delivery show distinct single-cell characteristics. A simple two-step stochastic rate model can reproduce this.

Detection of mRNA

As gene expression studies gain increasing significance in drug discovery, translating these results into pharmacologically meaningful signature genes becomes increasingly essential. How these are interpreted in the context of metabolic or apoptotic pathways and how they can be related to preclinical models will have significant implications for using these results for clinical trials.

The study of cellular gene expression requires the use of fluorescent markers. One of the most popular is the enhanced green fluorescent protein (GFP), which produces bright green fluorescence with an emission peak of 509 nm. This product is a capped mRNA that has been polyadenylated and is optimized for mammalian systems. It mimics a fully processed mature mRNA and is suitable for transient labeling, RNA-Seq, and cell viability assays.

Compared to the delivery of plasmid DNA (pDNA), detecting eGFP mRNA yields single-cell expression time courses independent of the cell type and well described by mathematical modeling. This is partly because mRNA has higher translation efficiency than pDNA and can be stored in lipoplexes for weeks without loss of fluorescence.

In addition, the authors developed a slow tissue freezing protocol that allowed for much more robust and reproducible preservation of EGFP fluorescence in samples from the epididymis and testis. 

Detection of Protein

The most widely used reporter system for gene expression studies involves a functional protein fused to a green fluorescent protein (GFP). GFP fluorescence is measured in individual cells by flow cytometry, and the data are plotted as averages per population of cells. The method is exceptionally robust and reproducible and allows for the quantification of changes in transcriptional activity.

Flow cytometric measurement of eGFP expression is straightforward to execute and provides a compassionate means for assessing the effects of experimental manipulations. FC is also an exceptionally reliable method for measuring the variation of expression among groups of cells and for hypothesis testing.

Detection of DNA

By fusing a gene of interest to a fluorescent marker, the expression of that gene can be directly measured in live cells by fluorescence microscopy. The method can measure the tagged protein’s expression level and localization. This allows researchers to determine what part of the cell the protein is located within and whether or not it is interacting with other proteins.

By measuring GFP fluorescence in cells before flow cytometry, it is possible to determine the number of cells expressing the gene of interest. This makes distinguishing between transfected and non-transfected cells easy, allowing the data to be analyzed and the results to be reported.

As an alternative to detecting the mRNA in cells by fluorescent staining, it is also possible to see the mRNA as DNA using a polymerase chain reaction (PCR). This allows the results to be analyzed as both an increase and decrease in the amount of DNA present in a sample.

A limiting step in both mRNA and pDNA delivery is the endosomal uptake and degradation of the nucleic acid. As a result, it is generally accepted that the number of successfully delivered and translated mRNAs in a population of cells can be reproduced by a stochastic rate model. This allows researchers to predict the expected kinetics of expression and how much mRNA will likely be expressed in a given time frame.

Detection of RNA

The eGFP mRNA is a commonly used gene expression control in mammalian cell culture. It encodes a fluorescent protein that produces a green light at excitation/emission wavelengths of 488 nm/509 nm. It is a valuable tool for testing transfection reagents because it is more sensitive than DNA and expresses a stable protein within cells. It is also a useful tool for monitoring dynamic transcriptional activity. A clever approach has been developed to detect dynamic changes in transcription through the ratiometric detection of short-lived dGFP/long-lived dRFP produced by a self-cleaving 2A peptide between eGFP and RFP.

To examine the effectiveness of mRNA electroporation in vivo, the mRNA encoding enhanced green fluorescent protein (EGFP) was electroporated into the lateral ventricle of embryonic (E)13.5 mouse brains. The EGFP fluorescence was observed by confocal microscopy, and the signal was monitored over time. The results showed that the EGFP fluorescence was detected 1 h after electroporation, reached a plateau 6 h after electroporation, and rapidly declined.

These data were fitted according to a kinetic rate model, and the distributions of the onset times and the expression rates kTL * m0. The held onset times t0 and t1 correspond to the mRNA accumulated in endosomal compartments. The results demonstrate that mRNA can efficiently be delivered to the cell and expressed similarly to pDNA.

*This is a collaboration post