An adapted protocol to overcome endosomal damage caused by polyethylenimine (PEI) mediated transfections.

Abstract

Transfection of cells using polyethylenimine (PEI) polymers is a widely used technique for gene insertion and exogenous protein expression that offers several advantages over virus or lipid-based transfection methods. PEI facilitates endocytosis of DNA into the cell, release of DNA from the endolysosomal system and protection from nucleases. Although PEI is extensively investigated for application in research and therapy, the mechanism of its release from the endosomal system has remained a point of debate. The most prevailing model states that PEI causes endosomal rupture, resulting in release of PEI-DNA complexes into the cytosol. Hence, the use of PEI transfection should be applied with great care in studies on the endosomal system. Here we study the effect of PEI transfections on the endolysosomal system. Using fluorescent and electron mi-croscopy we find a decrease in early endosomes after PEI transfection. Adapting the PEI transfection protocol by including a chase time after initial uptake of the PEI-DNA polyplexes rescues this phenotype without affecting transfection efficiency. Our data result in an adapted protocol for PEI transfection that can be used in studies involving the endolysosomal system.