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

Publication ,  Journal Article
Wu, M; Yuan, F
Published in: Plos One
January 2011

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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Published In

Plos One

DOI

EISSN

1932-6203

ISSN

1932-6203

Publication Date

January 2011

Volume

6

Issue

6

Start / End Page

e20923

Related Subject Headings

  • Transfection
  • Plasmids
  • Mice
  • Kinetics
  • Intracellular Space
  • General Science & Technology
  • Gene Expression Regulation
  • Endocytosis
  • Electricity
  • Dynamins
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Wu, M., & Yuan, F. (2011). Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells. Plos One, 6(6), e20923. https://doi.org/10.1371/journal.pone.0020923
Wu, Mina, and Fan Yuan. “Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells.Plos One 6, no. 6 (January 2011): e20923. https://doi.org/10.1371/journal.pone.0020923.
Wu, Mina, and Fan Yuan. “Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells.Plos One, vol. 6, no. 6, Jan. 2011, p. e20923. Epmc, doi:10.1371/journal.pone.0020923.

Published In

Plos One

DOI

EISSN

1932-6203

ISSN

1932-6203

Publication Date

January 2011

Volume

6

Issue

6

Start / End Page

e20923

Related Subject Headings

  • Transfection
  • Plasmids
  • Mice
  • Kinetics
  • Intracellular Space
  • General Science & Technology
  • Gene Expression Regulation
  • Endocytosis
  • Electricity
  • Dynamins