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

Wu M, Yuan F - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

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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Reduction in cellular uptake of pDNA and the eTE by endocytic inhibitor treatment.pDNA covalently labeled with rhodamine (red) was electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) into cells pre-treated with (A) DMSO (drug vehicle) or (B) dynasore (80 µM) for 1 hr. After electrotransfection, the cells were incubated at 37°C to enable cellular uptake of pDNA for 30 min. At the end of incubation, the cells were examined using confocal microscopy. Arrows in the microscopic images denote pDNA internalized by cells. To visualize three-dimensional distribution of pDNA in the cytosol, two optical cross-sections of DMSO-treated cells in x–z and y–z planes are shown in Panel (C). Effects of endocytic inhibitor treatment on the eTE are shown in Panel (D). Cells were treated with DMSO (Ctrl), 28 µM CPZ, 200 µM genistein (GE), or 80 µM dynasore (DN) for 1 hr prior to electrotransfection with the GFP-encoding pDNA. The eTE, defined as the percent of live cells expressing GFP, was quantified after cells were cultured at 37°C for 24 hr. n = 4–6. * P<0.05 and ** P<0.005 (Mann-Whitney U test).
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pone-0020923-g004: Reduction in cellular uptake of pDNA and the eTE by endocytic inhibitor treatment.pDNA covalently labeled with rhodamine (red) was electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) into cells pre-treated with (A) DMSO (drug vehicle) or (B) dynasore (80 µM) for 1 hr. After electrotransfection, the cells were incubated at 37°C to enable cellular uptake of pDNA for 30 min. At the end of incubation, the cells were examined using confocal microscopy. Arrows in the microscopic images denote pDNA internalized by cells. To visualize three-dimensional distribution of pDNA in the cytosol, two optical cross-sections of DMSO-treated cells in x–z and y–z planes are shown in Panel (C). Effects of endocytic inhibitor treatment on the eTE are shown in Panel (D). Cells were treated with DMSO (Ctrl), 28 µM CPZ, 200 µM genistein (GE), or 80 µM dynasore (DN) for 1 hr prior to electrotransfection with the GFP-encoding pDNA. The eTE, defined as the percent of live cells expressing GFP, was quantified after cells were cultured at 37°C for 24 hr. n = 4–6. * P<0.05 and ** P<0.005 (Mann-Whitney U test).

Mentions: Three pharmacological inhibitors of endocytosis were used in the study: chlorpromazine (CPZ), genistein, and dynasore, which block clathrin-coated pit formation, caveolae-mediated endocytosis, and dynamin activity, respectively [34], [35], [36]. Dynamin, a GTPase involved in clathrin-mediated as well as certain clathrin-independent endocytosis, facilitates fission of vesicles from the plasma membrane, resulting in release of the vesicles into the cytosol [37]. In the first experiment, B16.F10 cells were treated with dynasore or the drug vehicle DMSO (control) for 1 hr prior to electrotransfection with rhodamine-labeled pDNA. After electrotransfection, cells were incubated at 37°C for 30 min and the internalized pDNA molecules were visualized using confocal microscopy. Figures 4A through 4C demonstrated qualitatively that dynasore treatment could reduce the uptake and intracellular distribution of electrotransfected pDNA in cells. In the second experiment, B16.F10 cells were pre-treated with each of the pharmacological inhibitors for 1 hr followed by electrotransfection with unlabeled pDNA encoding GFP. The eTE was quantified after 24 hr of incubation. The data shown in Figure 4D demonstrated that all three pharmacological inhibitors could significantly reduce eTE (P<0.05), compared to the control group, in which the cells were treated with equivalent volumes of DMSO alone.


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

Wu M, Yuan F - PLoS ONE (2011)

Reduction in cellular uptake of pDNA and the eTE by endocytic inhibitor treatment.pDNA covalently labeled with rhodamine (red) was electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) into cells pre-treated with (A) DMSO (drug vehicle) or (B) dynasore (80 µM) for 1 hr. After electrotransfection, the cells were incubated at 37°C to enable cellular uptake of pDNA for 30 min. At the end of incubation, the cells were examined using confocal microscopy. Arrows in the microscopic images denote pDNA internalized by cells. To visualize three-dimensional distribution of pDNA in the cytosol, two optical cross-sections of DMSO-treated cells in x–z and y–z planes are shown in Panel (C). Effects of endocytic inhibitor treatment on the eTE are shown in Panel (D). Cells were treated with DMSO (Ctrl), 28 µM CPZ, 200 µM genistein (GE), or 80 µM dynasore (DN) for 1 hr prior to electrotransfection with the GFP-encoding pDNA. The eTE, defined as the percent of live cells expressing GFP, was quantified after cells were cultured at 37°C for 24 hr. n = 4–6. * P<0.05 and ** P<0.005 (Mann-Whitney U test).
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Related In: Results  -  Collection

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pone-0020923-g004: Reduction in cellular uptake of pDNA and the eTE by endocytic inhibitor treatment.pDNA covalently labeled with rhodamine (red) was electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) into cells pre-treated with (A) DMSO (drug vehicle) or (B) dynasore (80 µM) for 1 hr. After electrotransfection, the cells were incubated at 37°C to enable cellular uptake of pDNA for 30 min. At the end of incubation, the cells were examined using confocal microscopy. Arrows in the microscopic images denote pDNA internalized by cells. To visualize three-dimensional distribution of pDNA in the cytosol, two optical cross-sections of DMSO-treated cells in x–z and y–z planes are shown in Panel (C). Effects of endocytic inhibitor treatment on the eTE are shown in Panel (D). Cells were treated with DMSO (Ctrl), 28 µM CPZ, 200 µM genistein (GE), or 80 µM dynasore (DN) for 1 hr prior to electrotransfection with the GFP-encoding pDNA. The eTE, defined as the percent of live cells expressing GFP, was quantified after cells were cultured at 37°C for 24 hr. n = 4–6. * P<0.05 and ** P<0.005 (Mann-Whitney U test).
Mentions: Three pharmacological inhibitors of endocytosis were used in the study: chlorpromazine (CPZ), genistein, and dynasore, which block clathrin-coated pit formation, caveolae-mediated endocytosis, and dynamin activity, respectively [34], [35], [36]. Dynamin, a GTPase involved in clathrin-mediated as well as certain clathrin-independent endocytosis, facilitates fission of vesicles from the plasma membrane, resulting in release of the vesicles into the cytosol [37]. In the first experiment, B16.F10 cells were treated with dynasore or the drug vehicle DMSO (control) for 1 hr prior to electrotransfection with rhodamine-labeled pDNA. After electrotransfection, cells were incubated at 37°C for 30 min and the internalized pDNA molecules were visualized using confocal microscopy. Figures 4A through 4C demonstrated qualitatively that dynasore treatment could reduce the uptake and intracellular distribution of electrotransfected pDNA in cells. In the second experiment, B16.F10 cells were pre-treated with each of the pharmacological inhibitors for 1 hr followed by electrotransfection with unlabeled pDNA encoding GFP. The eTE was quantified after 24 hr of incubation. The data shown in Figure 4D demonstrated that all three pharmacological inhibitors could significantly reduce eTE (P<0.05), compared to the control group, in which the cells were treated with equivalent volumes of DMSO alone.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

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.

Show MeSH
Related in: MedlinePlus