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dc.contributor.author Wu, M
dc.contributor.author Yuan, F
dc.coverage.spatial United States
dc.date.accessioned 2011-06-30T18:48:11Z
dc.date.issued 2011
dc.identifier http://www.ncbi.nlm.nih.gov/pubmed/21695134
dc.identifier PONE-D-11-05519
dc.identifier.citation PLoS One, 2011, 6 (6), pp. e20923 - ?
dc.identifier.uri http://hdl.handle.net/10161/4640
dc.description.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.
dc.description.sponsorship This work was supported in part by a grant from the National Institutes of Health (NIH; CA94019). MW was supported in part by a NIH training grant for the Center for Biomolecular and Tissue Engineering at Duke University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. en_US
dc.format.extent e20923 - ?
dc.language eng
dc.language.iso en_US en_US
dc.relation.ispartof PLoS One
dc.relation.isversionof 10.1371/journal.pone.0020923
dc.subject Adsorption
dc.subject Animals
dc.subject Cations, Divalent
dc.subject Cell Line, Tumor
dc.subject Cell Membrane
dc.subject DNA
dc.subject Dynamins
dc.subject Electricity
dc.subject Endocytosis
dc.subject Gene Expression Regulation
dc.subject Intracellular Space
dc.subject Kinetics
dc.subject Mice
dc.subject Plasmids
dc.subject Transfection
dc.title Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells.
dc.type Journal Article
duke.contributor.id mw57 en_US
duke.contributor.id fyuan en_US
pubs.author-url http://www.ncbi.nlm.nih.gov/pubmed/21695134
pubs.issue 6
pubs.organisational-group /Duke
pubs.organisational-group /Duke/Pratt School of Engineering
pubs.organisational-group /Duke/Pratt School of Engineering/Biomedical Engineering
pubs.organisational-group /Duke/School of Medicine
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments/Ophthalmology
pubs.organisational-group /Duke/School of Medicine/Institutes and Centers
pubs.organisational-group /Duke/School of Medicine/Institutes and Centers/Duke Cancer Institute
pubs.publication-status Published
pubs.volume 6
dc.identifier.eissn 1932-6203

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