Fluorescent proteins induce oxidative stress by catalytically generating superoxide anion free radical (O2*-) and hydrogen peroxide (H2O2) independently of NAD(P)H during their maturation. However, eGFP generates H2O2 levels below the cells’ baseline level and does not cause lethality.
How eGFP mRNA Transforms Cells
Green fluorescent protein (GFP) is a viral marker for gene expression studies. Light microscopy quickly observes GFP fluorescence, allowing a direct correlation of changes in reporter gene expression with intracellular accumulation of the protein. This quantitative, reliable reporter assay is particularly well suited for flow cytometric analysis because it provides a clear and straightforward measure of protein accumulation independent of changes in cell density or dissociation procedures.
The GFP coding sequence was derived from the jellyfish Aequorea victoria, producing bright green fluorescence with an emission peak at 509 nm. Vernal Biosciences eGFP mRNA is codon optimized for mammalian systems and is polyadenylated to mimic fully processed mature mRNA. It is capped using a proprietary co-transcriptional capping method.
GFP mRNA can also be delivered to cells through lipid nanoparticles (LNP). The LNP encapsulates the mRNA for optimal delivery and stability. eGFP-LNP is a valuable tool for assays of RNA delivery and translation efficiency, cell viability, and other parameters affected by cellular conditions. Moreover, eGFP-LNP can generate a line of transgenic animals that exhibit a specific genetically encoded response to physiological stimuli or disease conditions.
How eGFP mRNA Transforms Proteins
The expression of GFP mRNA in cells forms a green fluorescent protein (GFP). The GFP fluorescence signal can be used to monitor changes in gene expression. In addition, GFP can be used as a marker for protein localization in living cells. For example, a GFP mRNA expression construct can identify cell regions where Cajal bodies are present. A GFP mRNA expression construct can also identify the sites where spliceosomal nitric oxide synthase is located.
GFP mRNA encodes an enhanced version of the green fluorescent protein, originally isolated from the jellyfish Aequorea Victoria, that yields bright green fluorescence with an emission peak at 509 nm. EGFP is a direct detection reporter gene in mammalian cell culture commonly used as a positive control for transfection experiments.
A mutated form of EGFP has been developed to be brighter and more acid resistant than the wild-type EGFP protein. A mutant of EGFP engineered to have a reduced tendency to dimerize is commercially available.
The eGFP mRNA is capped using a proprietary co-transcriptional capping method and polyadenylated to mimic a fully processed mature mRNA. This mRNA is optimized for use in mammalian systems and contains the codon-optimized nucleotide sequence of EGFP.
How eGFP mRNA Transforms DNA
GFP (enhanced green fluorescent protein) is one of the most famous reporter genes for cellular imaging. It is bright, stable, and non-toxic when illuminated inside living cells. It has opened new opportunities for analyzing complex cellular processes previously impossible to study with fixed specimens.
In addition to its intrinsic fluorescence, GFP is also a valuable tool for detecting the presence of genotoxic lesions in DNA. Cessation of transcription occurs rapidly when a lesion blocks the polymerases that synthesize mRNA from the template DNA strand. GFP is beneficial for this purpose because it binds to the same polymerases targeted by the RNA helicase ERN1 for degradation.
To determine whether ERN1 targets the EGFP mRNA for degradation, we introduced a silent mutation into the coding sequence of EGFP. The mutation replaces glutamine with adenine at the 552nd position of the mRNA encoding region. As expected, this mutation eliminates the ERN1 target site and renders mRNA resistant to ERN1-dependent degradation. Nevertheless, mRNA levels of conventional EGFP and the EGFPG552A mutant are reduced upon BafA1 treatment.
Because eGFP is non-toxic, its measurement in individual cells is ideal for examining gene expression kinetics with flow cytometry. Assuming that cell populations are otherwise identical, the ability to analyze the level of GFP accumulation in each cell can be beneficial in assessing hypothesis testing and discriminating between experimental groups.
How eGFP mRNA Transforms RNA
GFP is a bright green fluorescent protein derived from the jellyfish Aequorea victoria. When exposed to blue light, the chromophore of GFP emits green fluorescence at a readily detectable wavelength. GFP is commonly used as a direct detection reporter in cells because it is much less toxic to living organisms than other small fluorescent molecules such as fluorescein isothiocyanate (FITC).
The presence of GFP within a living cell provides investigators with several valuable tools. For example, a GFP-tagged protein can monitor the interaction of two proteins in a living organism. This can be done using the FRET technique, which uses donor and acceptor pairs of complementary fluorescent proteins to detect when they are close together (within 10 nm).
EGFP also serves as a convenient marker for the fluid-phase uptake of proteins.
Another valuable application for eGFP mRNA is the creation of fusion proteins in which it is fused to a protein of interest. Time-lapse imaging techniques can then visualize these fusions in a living organism.
