Mitosis enhances transgene expression of plasmid delivered by cationic liposomes

In this study, the effect of mitosis on transgene expression was examined by quantitative flow cytometry. Transfection of HeLa cells synchronized at late GI phase or G2/M phase was performed using a liposomal vector containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dioleoyl-phosphatidylethanolamine (DOPE) (1:1 mol/mol). Cell samples were transfected and subsequently maintained in G1 phase for various durations to modulate the time between plasmid entry and mitosis. The plasmid contains the sequence for a mutated green fluorescent protein (GFP(S65T)) that was used to examine transgene expression. Ethidium monoazide (EMA) labeled plasmid was employed to examine the association of plasmid with the cell membrane. The percentage of cells expressing GFP(S65T) increased sharply as the synchronized cell population passed through M phase, suggesting that an event associated with mitosis is essential for transgene expression. Expression levels of the transgene then declined 18 h after mitosis irrespective of transfection strategy. All transfection strategies resulted in the same maximum percentage of GFP(S65T) positive cells (40%) and average GFP(S65T) expression level (3.14×10⁶ molecules per positive cell). Association of plasmid with the cell membrane at late G1 phase was 1.5 fold of that at G2/M phase. These data are evidence for control of transgene expression triggered by events associated with cell cycle