In order for the human body to function, billions of cells must collaborate. Examining the physical collaborations between these cells is imperative for grasping cell activity in both illness and health.
G-baToN relies on surface GFP expression in ‘Sender’ cells to rapidly label ‘Receiver’ cells upon contact. This system broadly applies to various primary cells and heterotypic cell-cell interactions, including endothelial cells interacting with smooth muscle cells and astrocytes interacting with neurons.
How eGFP mRNA is made
eGFP (enhanced green fluorescent protein) is a versatile genetic marker for monitoring protein synthesis in individual living cells. It is a handy tool in neuroscience because its fluorescence is readily detectable at relatively low concentrations (5 M) in the brain, and it can be excited at the emission maximum of 507 nm.
EGFP contains a coding sequence that is codon-optimized for expression in mammalian cells. Moreover, the mRNA for eGFP contains a 32-nucleotide iron-responsive element (IRE) that can bind to specific cellular proteins that mediate translation. This IRE site is located at the end of the coding region, and its deletion reduces functional mRNA half-life.
The IRE of eGFP mRNA also acts as an acceptor for adenosine triphosphate, a small molecule that can promote translation in high levels of tetracyclic uracil nucleotides. This process is mediated by a complex network of proteins that regulates the levels and distribution of UTP in the cell.
Various protein engineering techniques have yielded several variants of EGFP. It is also possible to engineer FPs with improved fluorescence properties by introducing point mutations that alter the chromophore-forming amino acids.
eGFP mRNA Applications
eGFP provides a valuable tool to observe the fate of mRNA during delivery and expression. By fusing the eGFP gene with a gene of interest, protein synthesis is maintained while acquiring a fluorescent property, allowing researchers to observe the localization and destiny of the resulting proteins.
eGFP has many applications for visualizing cellular processes in ex vivo tissue. It is often used to study protein trafficking, cellular dynamics, cell division, migration, and other processes. In addition, eGFP can be incorporated into cell organelles, allowing Vernal Biosciences researchers to monitor the function of specific subunits or enzymes during protein expression.
Using various intradermal injection techniques, Cy5 labeled mRNA and eGFP expression was monitored in skin sections obtained from ex vivo porcine skin samples with a confocal microscope equipped with a 63x oil objective. The difference in the spatial distributions of the mRNA and GFP signals identified injection sites.
Interestingly, despite differences in the delivery modalities, the overall mRNA distribution and expression trend were similar in all delivery methods tested. In the epidermis, eGFP was expressed away from the injection site by epidermal keratinocytes and by epithelial cells in appendageal structures such as PSU and sweat glands, and to a lesser extent by dermal fibroblasts.
eGFP mRNA Transfection
eGFP mRNA is a messenger RNA that encodes for an enhanced green fluorescent protein (EGFP). Unlike traditional mRNA, eGFP mRNA has a 5′ cap structure. This increases the stability of mRNA and improves translation efficiency. In addition to enhancing translation efficiency, the cap structure prevents degradation of mRNA by enzymes in the cell.
mRNA transfection is an essential step in the process of gene expression.
To demonstrate the high transfection efficiency of the TransIT-mRNA Reagent, eGFP mRNA was transfected into various cell types. BJ fibroblasts, HEK293 cells, and ECs were transfected with varying amounts of eGFP mRNA. The amount of GFP expressed in the cells was determined using flow cytometry. It was found that 0.5 mg of mRNA resulted in a significant percentage of eGFP-positive cells.
mRNA transfections were also performed in murine primary bone marrow-derived dendritic cells (BMDCs) and mouse dendritic cell types JAWS II and DC 2.4. The mRNA was capped and polyadenylated with a 5′ oligo-T tail of 120 thymidines. It was then inserted into a plasmid that contains the ura1 gene of Schizophyllum commune as a selectable marker. The resulting plasmids were then transformed into the Ural1 auxotrophic strain of P. chrysosporium and assayed for GFP fluorescence.
eGFP mRNA Expression
The EGFP gene produces a protein that emits bright green light upon excitation at 509 nm. This protein is often used as a reporter for observing the movement and localization of other proteins within the cell. EGFP is a straightforward and convenient tool for analyzing various aspects of cellular function.
Using this tool, you can quickly and easily determine if your cell can process a specific substrate or perform a particular activity. This can also be a helpful way to test the effectiveness of specific compounds or antibodies that target an activity, such as autophagy.
eGFP expression was determined 1, 2, and 3 days after transfection of cells with modified mRNA.
After exogenous mRNA delivery, all cell types produced functional eGFP, and the expression was maintained over three days. This information can be used to optimize experimental conditions so that each cell type can be successfully induced to produce desired proteins.