Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells.

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2011

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Abstract

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.

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10.1371/journal.pone.0020923

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Wu, Mina, and Fan Yuan (2011). Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells. PLoS One, 6(6). p. e20923. 10.1371/journal.pone.0020923 Retrieved from https://hdl.handle.net/10161/4640.

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Scholars@Duke

Yuan

Fan Yuan

Professor of Biomedical Engineering

Dr. Yuan has extensive experiences in analysis of therapeutic agent transport in mammalian cells, tissues, and organs, and development of effective strategies, design principles, and new technologies that can be used to facilitate the transport. The goal of his research is to improve delivery of therapeutic agents to their targets, which is crucial in treatment and prevention of diseases. He has published >100 scientific papers in peer-reviewed journals, and a textbook on transport analysis in biological systems that has been used to teach undergraduate and graduate courses in many universities.


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