mRNA Transfection of Endothelial Cells with RJH Transfection Reagents
Delivering messenger RNA (mRNA) is a new approach for rapid expression and secretion of proteins in human cells. It is providing a more effective alternative to plasmid DNA based expression systems since it may not be necessary to target the mRNA to the nucleus; simply delivering the mRNA to cytoplasm might be sufficient for robust expression. Transfection reagents are actively developed for this application. Developing effective transfection regions for delivering mRNA to endothelial cells is attractive since these cells line up the blood vessels and it is the first cell type that comes in contact with agents administered in the body. The cells also control a plethora of physiological activities in the body, so that they constitute an important cell type for scientific investigations and therapeutic purposes. Below is a summary of recent experience with the use of RJH reagents for modification of human endothelial cells with mRNA.
Human breast tumor-associated endothelial cells were purchased from Cell Biologics (Cat No HC-6020; Chicago, IL) and seeded in H1168 Complete Human Endothelial Cell Medium (Cell Biologics) in multiwell plates 1 day before transfection. The transfection was carried out with a modified mRNA (details on mRNA structure withheld due to confidentiality) by using the transfection reagents Prime-Fect (RJH Biosciences), LipofectamineTM 3000 (ThermoFisher) and other experimental reagents from the RJH Biosciences. The complexes were formulated at a reagent:nucleic acid ratio of 5:1 (w/w). After incubation of cells with the complexes for 2 days, the media were replaced, the cells were washed and recovered (by trypsin) for flow cytometry analysis. The mean percentage of cells positive for GFP expression was summarized below.
The results from flow cytometry analysis are shown on the left as the percentage of cells expressing GFP. Prime-Fect was effective for mRNA delivery but other reagents currently under development by RJH Biosciences were even more effective, clearly superseding the performance of the LipofectamineTM 3000.
| Data courtesy of Dr. Xin Liu, Shanghai 9th People’s Hospital (China)