Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells.
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Electric field mediated gene delivery or electrotransfection is a widely used method in various studies ranging from basic cell biology research to clinical gene therapy. Yet, mechanisms of electrotransfection are still controversial. To this end, we investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane and how treatment of cells with three endocytic inhibitors (chlorpromazine, genistein, dynasore) or silencing of dynamin expression with specific, small interfering RNA (siRNA) would affect the eTE. Our data demonstrated that the presence of divalent cations (Ca(2+) and Mg(2+)) in electrotransfection buffer enhanced pDNA adsorption to cell membrane and consequently, this enhanced adsorption led to an increase in eTE, up to a certain threshold concentration for each cation. Trypsin treatment of cells at 10 min post electrotransfection stripped off membrane-bound pDNA and resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake of pDNA (between 10 and 40 min) far exceeded the lifetime of electric field-induced transient pores (∼10 msec) in the cell membrane. Furthermore, treatment of cells with the siRNA and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. These findings suggest that electrotransfection depends on two mechanisms: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.
Cell Line, Tumor
Gene Expression Regulation
Published Version (Please cite this version)10.1371/journal.pone.0020923
Publication InfoWu, Mina; & Yuan, Fan (2011). Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells. PLoS One, 6(6). pp. e20923. 10.1371/journal.pone.0020923. Retrieved from https://hdl.handle.net/10161/4640.
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Professor of Biomedical Engineering
Dr. Yuan's research interests include drug and gene delivery, mechanisms of molecular transport in cells and tissues, and tumor pathophysiology. Cure of cancer through chemotherapy requires drug molecules to reach all tumor cells at an adequately high concentration. At present, such a requirement cannot be satisfied in most patients. This is because (a) amount of drugs that can be administered into patients is limited by normal tissue tolerance and (b) drug distribution and cellular r