Jellyfish are inarguably one of nature’s most extraordinary creatures. But their life cycles and behavior remain a mystery.
With some genetic tinkering, scientists have discovered how to read the neurons in a tiny species of see-through jellyfish. They watched as the neurons worked together to perform complex autonomous movements, like transferring shrimp from tentacles to mouth.
What is eGFP mRNA?
Green fluorescent protein (GFP) is a small molecule that binds to a specific site in the mammalian genome and induces the translation of a protein that emits bright green light. GFP is commonly used as a reporter gene for mammalian cells and tissue detection. The gene coding for GFP can also be fused to other proteins to create various useful biochemical tools.
eGFP mRNA can be transfected into mammalian cells to produce GFP, easily detectable by fluorescence microscopy or flow cytometry. It can also be incorporated into lipid nanoparticles (LNP) to deliver eGFP mRNA directly to cells without needing an additional transfection.
The amino acid sequences of EGFP, VFP, and Venus were retrieved from the EMBL nucleotide and protein sequence databases. Site-directed mutagenesis was performed using the kit to introduce N158K and T160R mutations in the VFP chromophore-forming residues. The mutants were further characterized by using fluorescence correlation spectroscopy (FCS).
How was eGFP mRNA discovered?
It was a fantastic moment in scientific history when researchers realized that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria could be used as a marker to track proteins in living cells directly. GFP glows bright green when exposed to ultraviolet light, so it’s easy to spot if it’s present in a cell. By tagging GFP to other proteins and watching them interact inside a cell, scientists could see never before seen processes happening in real time.
GFP’s chromophore is a cage-like structure resembling the backbone of a DNA molecule. The structure shields the chromophore from jostling water molecules, which would typically rob it of energy once it absorbs a photon. Instead, the chromophore releases the energy as a fluorescent signal. GFP’s brightness, photostability, and monomeric character make it one of the most popular tools in contemporary bioscience.
In addition, using GFP in fusion proteins provides another valuable tool for tracking proteins in live cells. Scientists have used GFP fusion proteins to study the structure and dynamics of various organisms, from bacteria and yeast to mammalian cells and even viruses.
eGFP can be easily tethered to any other protein, and the result is an exciting new method of imaging cellular processes in real-time. The sensitivity of eGFP allows for detecting very low protein levels, such as viral particles or enzymes, and is helpful in a wide range of applications.
What are the implications of eGFP mRNA?
GFP mRNA allows scientists to track the expression of genes in intact cells and can be used for various applications. In addition, eGFP can be used as a marker to select and enrich gene-modified cells for flow cytometry. However, it is essential to note that GFP+ cells can trigger T-cell immune responses due to the major histocompatibility complex’s processing and presentation of the transgene-derived peptides.
Moreover, the use of eGFP may be limited by the fact that eGFP expression is regulated by a lysosomal degradative enzyme, BafA1. This enzyme interacts with the 552nd base of the EGFP coding sequence, replacing it with an adenine (CAACAG) which does not affect the encoded amino acid. As such, the protein may be more acid resistant and brighter than wild-type EGFP when treated with BafA1 (Figure 2A).
To improve the brightness of GFP, the sequence has been modified to eliminate a putative ERN1 binding site and to increase its foldability at 37°C by replacing glutamine with an adenine (Emerald) [1, 11]. The resulting Emerald FP is significantly more acid resistant, brighter, and less sensitive to aggregation.
What is the future of eGFP mRNA?
The glow-in-the-dark protein that Osamu Shimomura and his team first stumbled upon in the 1960s has become a powerful tool for scientists studying everything from bacteria to human cells. Scientists now use eGFP to identify proteins in cells, visualize cell structures, and even track the movement of tumor cells in the body.
GFP isn’t the only fluorescent marker researchers can use, but it remains memorable because of its stability and brightness. It also “glows” on its own, without the help of chemicals or enzymes.
Jellyfish and comb jellies are among the few creatures on earth that produce their light, an ability known as bioluminescence. It still needs to be fully understood why these animals glow, but the bright flashes may serve as a warning to predators or attract prey.
But the real revolution that GFP has unleashed is its utility in genetic engineering. Scientists can now add a glowing tag to almost any gene, making it possible to study the behavior of the protein as it is expressed in living cells.
While the colorless version of eGFP is a standard, new versions with different colors and spectral properties continue to be developed. These new variants allow researchers to study different functions in the same cell by labeling only the most essential parts of a system. For example, scientists recently added a potassium channel to a GFP and found that the protein could illuminate a particular membrane potential in an organelle.