Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of trypsin treatment on pDNA adsorption to cell membrane and eTE.(A) YOYO 1-labeled pDNA (green) formed complexes with FM4-64FX labeled plasma membrane (red) following exposure of cells to pulsed electric field (400 V/cm, 5 msec, 8 pulses, 1 Hz). The image was taken shortly after the application of electric field. (B) The experimental protocol was the same as that in the Panel (A), except that at 10 min post electric field exposure, the cells were treated with 0.25% trypsin-EDTA solution for 30 min at 37°C. The image was taken after the trypsin treatment. (C) B16-F10 cells in pDNA solution were exposed to the same electric pulses (EP) as above. At 10 or 40 min post EP exposure, the cells were treated with 0.25% trypsin-EDTA for 30 min at 37°C. Then, the cells were cultured for 24 hr at 37°C. The eTE was measured as the percent of live cells expressing GFP and normalized by the data from the untreated group. The solid column and error bar represent mean and standard deviation of the relative eTE, respectively. n = 6–9. * P<0.05 (Mann-Whitney U test).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113837&req=5

pone-0020923-g003: Effects of trypsin treatment on pDNA adsorption to cell membrane and eTE.(A) YOYO 1-labeled pDNA (green) formed complexes with FM4-64FX labeled plasma membrane (red) following exposure of cells to pulsed electric field (400 V/cm, 5 msec, 8 pulses, 1 Hz). The image was taken shortly after the application of electric field. (B) The experimental protocol was the same as that in the Panel (A), except that at 10 min post electric field exposure, the cells were treated with 0.25% trypsin-EDTA solution for 30 min at 37°C. The image was taken after the trypsin treatment. (C) B16-F10 cells in pDNA solution were exposed to the same electric pulses (EP) as above. At 10 or 40 min post EP exposure, the cells were treated with 0.25% trypsin-EDTA for 30 min at 37°C. Then, the cells were cultured for 24 hr at 37°C. The eTE was measured as the percent of live cells expressing GFP and normalized by the data from the untreated group. The solid column and error bar represent mean and standard deviation of the relative eTE, respectively. n = 6–9. * P<0.05 (Mann-Whitney U test).

Mentions: Previous studies have shown that eTE can be significantly reduced in bacterial and Chinese hamster ovary (CHO) cells if they are treated with DNase within a few seconds or ∼1 min, respectively, of pulsed electric field application [33]. The observation suggests that, at these time points, either the cell membrane was still permeable to DNase resulting in degradation of internalized pDNA, or a large fraction of pDNA molecules was not internalized shortly after electric field treatment. To investigate when pDNA internalization was fully completed, we treated cells with DNase (10 U per µg pDNA for 30 min at 37°C) to digest extracellular YOYO 1-labeled pDNA or trypsin (0.25% for 30 min at 37°C) to cleave DNA-bound proteins associated with the membrane, at 10 or 40 min after electric field application. The DNase treatment was inadequate in digesting membrane-bound pDNA, as determined by fluorescence microscopy. However, pDNA bound to the membrane could be effectively removed by trypsin (see Figure 3A). Trypsin treatment was also able to significantly reduce eTE if administered at 10 min but had no effect at 40 min, when compared to the control (i.e., no trypsin case) (see Figure 3B), suggesting that the internalization of membrane-bound pDNA was completed between 10 and 40 min after electric field application.


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

Wu M, Yuan F - PLoS ONE (2011)

Effects of trypsin treatment on pDNA adsorption to cell membrane and eTE.(A) YOYO 1-labeled pDNA (green) formed complexes with FM4-64FX labeled plasma membrane (red) following exposure of cells to pulsed electric field (400 V/cm, 5 msec, 8 pulses, 1 Hz). The image was taken shortly after the application of electric field. (B) The experimental protocol was the same as that in the Panel (A), except that at 10 min post electric field exposure, the cells were treated with 0.25% trypsin-EDTA solution for 30 min at 37°C. The image was taken after the trypsin treatment. (C) B16-F10 cells in pDNA solution were exposed to the same electric pulses (EP) as above. At 10 or 40 min post EP exposure, the cells were treated with 0.25% trypsin-EDTA for 30 min at 37°C. Then, the cells were cultured for 24 hr at 37°C. The eTE was measured as the percent of live cells expressing GFP and normalized by the data from the untreated group. The solid column and error bar represent mean and standard deviation of the relative eTE, respectively. n = 6–9. * P<0.05 (Mann-Whitney U test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113837&req=5

pone-0020923-g003: Effects of trypsin treatment on pDNA adsorption to cell membrane and eTE.(A) YOYO 1-labeled pDNA (green) formed complexes with FM4-64FX labeled plasma membrane (red) following exposure of cells to pulsed electric field (400 V/cm, 5 msec, 8 pulses, 1 Hz). The image was taken shortly after the application of electric field. (B) The experimental protocol was the same as that in the Panel (A), except that at 10 min post electric field exposure, the cells were treated with 0.25% trypsin-EDTA solution for 30 min at 37°C. The image was taken after the trypsin treatment. (C) B16-F10 cells in pDNA solution were exposed to the same electric pulses (EP) as above. At 10 or 40 min post EP exposure, the cells were treated with 0.25% trypsin-EDTA for 30 min at 37°C. Then, the cells were cultured for 24 hr at 37°C. The eTE was measured as the percent of live cells expressing GFP and normalized by the data from the untreated group. The solid column and error bar represent mean and standard deviation of the relative eTE, respectively. n = 6–9. * P<0.05 (Mann-Whitney U test).
Mentions: Previous studies have shown that eTE can be significantly reduced in bacterial and Chinese hamster ovary (CHO) cells if they are treated with DNase within a few seconds or ∼1 min, respectively, of pulsed electric field application [33]. The observation suggests that, at these time points, either the cell membrane was still permeable to DNase resulting in degradation of internalized pDNA, or a large fraction of pDNA molecules was not internalized shortly after electric field treatment. To investigate when pDNA internalization was fully completed, we treated cells with DNase (10 U per µg pDNA for 30 min at 37°C) to digest extracellular YOYO 1-labeled pDNA or trypsin (0.25% for 30 min at 37°C) to cleave DNA-bound proteins associated with the membrane, at 10 or 40 min after electric field application. The DNase treatment was inadequate in digesting membrane-bound pDNA, as determined by fluorescence microscopy. However, pDNA bound to the membrane could be effectively removed by trypsin (see Figure 3A). Trypsin treatment was also able to significantly reduce eTE if administered at 10 min but had no effect at 40 min, when compared to the control (i.e., no trypsin case) (see Figure 3B), suggesting that the internalization of membrane-bound pDNA was completed between 10 and 40 min after electric field application.

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