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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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Dependence of electrotransfection efficiency on cation concentrations.eTE is defined as the percent of live cells expressing GFP. B16-F10 cells were electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) with unlabeled GFP-encoding pDNA in a transfection buffer. GFP expression was measured using flow cytometry after 24 hr incubation. (A) The low ionic strength medium supplemented with Ca2+ or Mg2+ at varying concentrations was used as the electrotransfection buffer. n = 7–8. The symbols and error bars denote means and standard deviations, respectively. The peak eTE value in each curve was significantly higher than those at both ends of the same curve (P<0.05). In Panels (B) and (C), OptiMEM was used as the electrotransfection buffer. After 20 min incubation post electrotransfection, the cells were re-suspended in the low ionic strength medium supplemented with either Ca2+ or Mg2+ at varying concentrations and treated again with the same electric field. The GFP expression was quantified at 24 hr. n = 4. The filled circles denote data from individual samples, the “x” symbol represents the mean of the samples at a given cation concentration, and the line represents the linear regression of the mean data. The mean value was statistically independent of the variation in Ca2+ and Mg2+ concentrations (P>0.05, Mann Whitney U test).
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pone-0020923-g002: Dependence of electrotransfection efficiency on cation concentrations.eTE is defined as the percent of live cells expressing GFP. B16-F10 cells were electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) with unlabeled GFP-encoding pDNA in a transfection buffer. GFP expression was measured using flow cytometry after 24 hr incubation. (A) The low ionic strength medium supplemented with Ca2+ or Mg2+ at varying concentrations was used as the electrotransfection buffer. n = 7–8. The symbols and error bars denote means and standard deviations, respectively. The peak eTE value in each curve was significantly higher than those at both ends of the same curve (P<0.05). In Panels (B) and (C), OptiMEM was used as the electrotransfection buffer. After 20 min incubation post electrotransfection, the cells were re-suspended in the low ionic strength medium supplemented with either Ca2+ or Mg2+ at varying concentrations and treated again with the same electric field. The GFP expression was quantified at 24 hr. n = 4. The filled circles denote data from individual samples, the “x” symbol represents the mean of the samples at a given cation concentration, and the line represents the linear regression of the mean data. The mean value was statistically independent of the variation in Ca2+ and Mg2+ concentrations (P>0.05, Mann Whitney U test).

Mentions: It has been reported that pulsed electric field induces complex formation between pDNA and cell membrane [22], [23], [24]. Although mechanisms of this complex formation are still unknown, we hypothesized that the process could be facilitated by divalent or multi-valent cations that cross-link and, hence, anchor pDNA to negatively charged carbohydrates and proteins on the plasma membrane. To test the hypothesis, we investigated the effects of divalent cations on pDNA binding to B16.F10 cell membrane, and quantified the dependence of pDNA adsorption on concentrations of Ca2+ and Mg2+. In the study, fluorescently labeled pDNA was mixed with cells suspended in various cation-supplemented buffers and left to incubate for 10 min on ice and 10 min at the room temperature to promote adsorption, before flow cytometric analysis (for details see the Materials and Methods section). It was observed that the percentage of pDNA-associated cells was a nonlinear function of the concentration for each cation (see Figure 1A), reaching a maximum value of ∼16%. The average intensity of the pDNA-associated cells, on the other hand, increased approximately linearly with no sign of saturation when the concentration of each cation was increased (see Figure 1B). To investigate the subsequent effects of this divalent cation-mediated pDNA adsorption on the electrotransfection of a reporter gene encoding green fluorescence protein (GFP), the cells were incubated with pDNA in the various cation-supplemented buffers for 10 min on ice to promote adsorption, followed by exposure to an electric field (8 pulses at 400 V/cm, 5 msec duration, and 1 Hz frequency) at the room temperature. After 24 hr of incubation, electrotransfection efficiency (eTE) was quantified using flow cytometry. The data shown in Figure 2A demonstrated that the eTE was enhanced by Ca2+ and Mg2+ at low concentrations but the enhancement was reduced when the concentrations were further increased. When no divalent cations were present in the low ionic strength buffer, the eTE was close to zero, suggesting that (a) cation-mediated pDNA adsorption was a necessary step for cell transfection and (b) electrophoresis of the polyanionic pDNA, which is inversely proportional to the ionic strength of a solution, did not play a critical role in electrotransfection, as also suggested by some previous studies [32]. To eliminate the possibility that the cation-dependent nature of eTE might be attributed to other cation-induced cellular effects aside from mediating pDNA-membrane interactions (i.e. changes in transcriptional and translational activities), a control experiment was performed in which cells were first electrotransfected with pDNA in OptiMEM. After a 20 min incubation period to permit sufficient membrane recovery, the cells were re-suspended in the low ionic strength buffer supplemented with cations at varying concentrations. The suspension was exposed again to the same electric field as before. The results shown in Figures 2B and 2C demonstrated that when cations were not present during the actual pDNA electrotransfection but rather, introduced into the cells in the presence of electric field 20 min later, the varying cation concentrations did not produce statistically significant changes in eTE, suggesting that Ca2+ and Mg2+ had minimal effects on normal transcriptional and translational activities in treated cells.


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

Wu M, Yuan F - PLoS ONE (2011)

Dependence of electrotransfection efficiency on cation concentrations.eTE is defined as the percent of live cells expressing GFP. B16-F10 cells were electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) with unlabeled GFP-encoding pDNA in a transfection buffer. GFP expression was measured using flow cytometry after 24 hr incubation. (A) The low ionic strength medium supplemented with Ca2+ or Mg2+ at varying concentrations was used as the electrotransfection buffer. n = 7–8. The symbols and error bars denote means and standard deviations, respectively. The peak eTE value in each curve was significantly higher than those at both ends of the same curve (P<0.05). In Panels (B) and (C), OptiMEM was used as the electrotransfection buffer. After 20 min incubation post electrotransfection, the cells were re-suspended in the low ionic strength medium supplemented with either Ca2+ or Mg2+ at varying concentrations and treated again with the same electric field. The GFP expression was quantified at 24 hr. n = 4. The filled circles denote data from individual samples, the “x” symbol represents the mean of the samples at a given cation concentration, and the line represents the linear regression of the mean data. The mean value was statistically independent of the variation in Ca2+ and Mg2+ concentrations (P>0.05, Mann Whitney U test).
© Copyright Policy
Related In: Results  -  Collection

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pone-0020923-g002: Dependence of electrotransfection efficiency on cation concentrations.eTE is defined as the percent of live cells expressing GFP. B16-F10 cells were electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) with unlabeled GFP-encoding pDNA in a transfection buffer. GFP expression was measured using flow cytometry after 24 hr incubation. (A) The low ionic strength medium supplemented with Ca2+ or Mg2+ at varying concentrations was used as the electrotransfection buffer. n = 7–8. The symbols and error bars denote means and standard deviations, respectively. The peak eTE value in each curve was significantly higher than those at both ends of the same curve (P<0.05). In Panels (B) and (C), OptiMEM was used as the electrotransfection buffer. After 20 min incubation post electrotransfection, the cells were re-suspended in the low ionic strength medium supplemented with either Ca2+ or Mg2+ at varying concentrations and treated again with the same electric field. The GFP expression was quantified at 24 hr. n = 4. The filled circles denote data from individual samples, the “x” symbol represents the mean of the samples at a given cation concentration, and the line represents the linear regression of the mean data. The mean value was statistically independent of the variation in Ca2+ and Mg2+ concentrations (P>0.05, Mann Whitney U test).
Mentions: It has been reported that pulsed electric field induces complex formation between pDNA and cell membrane [22], [23], [24]. Although mechanisms of this complex formation are still unknown, we hypothesized that the process could be facilitated by divalent or multi-valent cations that cross-link and, hence, anchor pDNA to negatively charged carbohydrates and proteins on the plasma membrane. To test the hypothesis, we investigated the effects of divalent cations on pDNA binding to B16.F10 cell membrane, and quantified the dependence of pDNA adsorption on concentrations of Ca2+ and Mg2+. In the study, fluorescently labeled pDNA was mixed with cells suspended in various cation-supplemented buffers and left to incubate for 10 min on ice and 10 min at the room temperature to promote adsorption, before flow cytometric analysis (for details see the Materials and Methods section). It was observed that the percentage of pDNA-associated cells was a nonlinear function of the concentration for each cation (see Figure 1A), reaching a maximum value of ∼16%. The average intensity of the pDNA-associated cells, on the other hand, increased approximately linearly with no sign of saturation when the concentration of each cation was increased (see Figure 1B). To investigate the subsequent effects of this divalent cation-mediated pDNA adsorption on the electrotransfection of a reporter gene encoding green fluorescence protein (GFP), the cells were incubated with pDNA in the various cation-supplemented buffers for 10 min on ice to promote adsorption, followed by exposure to an electric field (8 pulses at 400 V/cm, 5 msec duration, and 1 Hz frequency) at the room temperature. After 24 hr of incubation, electrotransfection efficiency (eTE) was quantified using flow cytometry. The data shown in Figure 2A demonstrated that the eTE was enhanced by Ca2+ and Mg2+ at low concentrations but the enhancement was reduced when the concentrations were further increased. When no divalent cations were present in the low ionic strength buffer, the eTE was close to zero, suggesting that (a) cation-mediated pDNA adsorption was a necessary step for cell transfection and (b) electrophoresis of the polyanionic pDNA, which is inversely proportional to the ionic strength of a solution, did not play a critical role in electrotransfection, as also suggested by some previous studies [32]. To eliminate the possibility that the cation-dependent nature of eTE might be attributed to other cation-induced cellular effects aside from mediating pDNA-membrane interactions (i.e. changes in transcriptional and translational activities), a control experiment was performed in which cells were first electrotransfected with pDNA in OptiMEM. After a 20 min incubation period to permit sufficient membrane recovery, the cells were re-suspended in the low ionic strength buffer supplemented with cations at varying concentrations. The suspension was exposed again to the same electric field as before. The results shown in Figures 2B and 2C demonstrated that when cations were not present during the actual pDNA electrotransfection but rather, introduced into the cells in the presence of electric field 20 min later, the varying cation concentrations did not produce statistically significant changes in eTE, suggesting that Ca2+ and Mg2+ had minimal effects on normal transcriptional and translational activities in treated cells.

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