Correlation between cationic lipid-based transfection and cell division

•Cationic lipid-based transfection supports protein expression without cell division.•Protein expression is unrelated to cell cycle status at the time of transfection.•Time-lapse imaging provides direct evaluation without statistical averaging.•Lipoplex dissociation is a likely target for improvement of transfection efficiency.

We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials.