Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells.
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.
Type
Journal articleSubject
AdsorptionAnimals
Cations, Divalent
Cell Line, Tumor
Cell Membrane
DNA
Dynamins
Electricity
Endocytosis
Gene Expression Regulation
Intracellular Space
Kinetics
Mice
Plasmids
Transfection
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https://hdl.handle.net/10161/4640Published Version (Please cite this version)
10.1371/journal.pone.0020923Publication Info
Wu, 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.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
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 analysi

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