Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization

Haberl, Saša; Miklavčič, Damijan; Pavlin, Mojca

doi:10.1016/j.bioelechem.2010.04.001

Cited by 35 publications

(37 citation statements)

References 51 publications

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“…Cell-impermeant molecules are then transported through these pores via mechanisms that may include diffusion [15], electrophoresis [3], and electroosmosis [16]. These mechanisms are likely to apply for delivery of small molecules but have yet to be shown to facilitate DNA transport across the membrane [12], [13], [16], [17], [18], [19], [20], [21]. More recently, emerging evidence from various studies is challenging the “electroporation” mechanism for gene delivery [22], [23], [24].…”

Section: Introductionmentioning

confidence: 99%

Membrane Binding of Plasmid DNA and Endocytic Pathways Are Involved in Electrotransfection of Mammalian Cells

Yuan

2011

PLoS ONE

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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 (Ca2+ and Mg2+) 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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Section: Introductionmentioning

confidence: 99%

Membrane Binding of Plasmid DNA and Endocytic Pathways Are Involved in Electrotransfection of Mammalian Cells

Yuan

2011

PLoS ONE

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“…Propidium iodide (PI) 2 µl of 0.15 mM was added to the suspension immediately before high voltage pulse application. After exposure to high voltage electric pulses, cells were incubated for 3 min at room temperature and then centrifuged for 5 min at 1000 rpm (984×g) [24]. The supernatant was removed and 200 µl of fresh media was added.…”

Section: Cell Electroporation and Propidium Iodide Uptakementioning

confidence: 99%

Assessment of the electrochemical effects of pulsed electric fields in a biological cell suspension

Chafai

Mehle

Tilmatine

et al. 2015

Bioelectrochemistry

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“…Several studies suggest that association with the cell membrane is required for entry into the cytoplasmic compartment of the cell. 44, 47, 55, 65, 145 However, microinjection and ballistic gene delivery bypass the cell membrane by directly transporting nucleic acids into the cell cytoplasm or the nucleus. Electroporation, sonoporation, and laser irradiation disrupt the cell membrane to facilitate infiltration of nucleic acids.…”

Section: Understanding Challenges That Limit Non-viral Gene Deliverymentioning

confidence: 99%

Physical Non-Viral Gene Delivery Methods for Tissue Engineering

2012

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The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

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Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization

Cited by 35 publications

References 51 publications

Membrane Binding of Plasmid DNA and Endocytic Pathways Are Involved in Electrotransfection of Mammalian Cells

Membrane Binding of Plasmid DNA and Endocytic Pathways Are Involved in Electrotransfection of Mammalian Cells

Assessment of the electrochemical effects of pulsed electric fields in a biological cell suspension

Physical Non-Viral Gene Delivery Methods for Tissue Engineering

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