Illuminating the Path to Advancements in Biotechnology
The eGFP gene produces bright green fluorescence with an emission maximum of 509 nm. This fluorescent protein can be used to identify and quantify transfected cells.
Gene expression changes can be monitored over time using a standard RT-qPCR assay. This assay identifies optimal conditions for mRNA delivery and the subsequent production of desired proteins.
The transfection efficiency depends on cell type, amount of mRNA, and transfection reagent. Consequently, optimizing the mRNA transfection process for each desired application is necessary.
Here, the mRNA transfection efficiency of BJ fibroblasts, ECs, and moDCs was evaluated by fluorescence microscopy and flow cytometry (FCM). Cell populations (3-7 x 105) were cultured in 48-well plates and transfected with various mRNA transfection complexes using the TransIT-mRNA Transfection Kit at different reagent-to-RNA ratios (1 ug/well). Afterward, EGFP expression was quantified by fluorescence microscopic analysis.
A high mRNA transfection efficiency was demonstrated for all three cell types, with ECs being the most efficient. The transfection efficiency was not enhanced by increasing the mRNA amount; even a low amount of mRNA was sufficient to induce eGFP expression in 75% of the cells.
To understand the impact of the mRNA poly(A) tail length, plasmids were generated with different poly(A) stretch sizes: one shorter than the control (76A-mRNA), one at the same size as the control (112A-mRNA), and one longer (148A-mRNA).
In addition, the effect of cap structure was investigated. EGFP expression in moDCs was assessed by FCM after transfection with IVT-mRNA capped with distinct cap analogs: Cap 0 (ARCA), Cap 1 (ARCA + MT and CleanCap), or no cap (Figure 4). No significant differences in eGFP production were observed in all three assays between the different cap structures, neither at low nor high mRNA doses.
GFP is a protein from the jellyfish Aequorea Victoria that produces bright green fluorescence. It is one of the most commonly used tools in research focusing on the transfection of genes to mammalian cells and tracking their expression. Vernal Biosciences eGFP mRNA is formulated at the optimal molar concentration for fast encapsulation and efficient mRNA delivery to mammalian cell cultures.
The ability to easily track gene and protein localization has opened new insights into cellular processes. Scientists use various methods to localize the expression of specific genes and proteins, including immunofluorescence microscopy, western blotting, and real-time PCR. In addition, fluorescent proteins can be tagged to identify specific cellular structures or processes. For example, a GFP-fused b actin allows researchers to examine the formation of actin filaments and their effect on cellular morphological changes and mechanical stiffness.
As researchers continue to move toward more physiologically relevant cell systems and to apply them in complex proteomic and genomic studies, the ability to efficiently transfect these cells becomes a critical requirement. While electroporation and viral-based methods can provide efficient delivery of DNA into cells, these technologies come with their challenges. Lipid-based and chemical reagents that perform well in various cell types are required to overcome these hurdles.
Traditional transfection reagents are typically one-component formulations of polyamines such as polyethyleneimine or cationic liposomes. They are designed to deliver a DNA complex into cells but do not typically control for the presence of other unintentional cellular effects.
Several gene expression profiling experiments were performed to understand transfection reagents’ mechanisms better. For these experiments, an EGFP-based chemiluminescent SEAP reporter gene was used as the target for all transfections. The 135 probes differentially expressed at 48 h post-transfection by all four transfection reagents were subjected to gene ontology enrichment analysis.
Several of these probes were found to be associated with an endonucleolytic activity of ERN1 (endoplasmic reticulum to nucleus signaling 1), which targets the RNA hairpin loop motif in EGFP mRNA. To determine if the ERN1 activity could be bypassed, a silent mutation was introduced into the 552nd base of the EGFP coding sequence. This substitution did not affect the encoded amino acid or disturb the local RNA structure and abolished the ERN1-dependent mRNA decay observed with conventional EGFP mRNA.
Introducing DNA, RNA, and proteins into cells allows researchers to manipulate cell behavior to study or leverage its function. This powerful tool has allowed for the development of many critical applications, such as gene therapy and induced pluripotent stem cell generation.
Viral delivery methods can achieve high transfection efficiencies in some cell types, but they are often laborious and must be carefully handled to avoid contamination or impact on functionality. In addition, they require expensive equipment and specialized reagents.
Chemical methods offer a gentler means of transfection, using cationic polymers such as DEAE-dextran or Polybrene to associate with negatively charged cell membranes to enable nucleic acid uptake. However, these chemical approaches typically have low efficiencies and can be cytotoxic to many cell types.
Lipid-based methods offer improved efficiencies and can be used in various biologically relevant cell types. However, lipid-based transfection is typically laborious and requires specific optimization for each cell type, which can be challenging given the wide variability in cellular characteristics across different species.
With the trend toward more physiologically relevant cell models, there is a growing need for efficient and simple transfection techniques. In the quest for more accurate and consistent results, developing new methods to achieve high transfection efficiencies with minimal impact on cell viability and function will be critical.