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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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Effects of dynamin II knockdown on pDNA electrotransfection.B16-F10 cells were transfected with either the control siRNA or one of the two specific siRNA oligos directed against two different sequences (i.e., Sq1 and Sq2) in mouse dynamin II gene for silencing its expression. The siRNA treatment was followed by a 48 hr incubation period prior to pDNA electrotransfection. Dynamin II and β-actin (loading control) expression levels in Western blot analysis are shown in Panel A and normalized electrotransfection efficiencies are shown in Panel B. The bars and error bars indicate the means and standard deviations of 4 independent trials, respectively. The data from each trial, used in mean and standard deviation calculation, was the average value of replicates or triplicates. *, P<0.05 (Mann-Whitney U test).
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pone-0020923-g005: Effects of dynamin II knockdown on pDNA electrotransfection.B16-F10 cells were transfected with either the control siRNA or one of the two specific siRNA oligos directed against two different sequences (i.e., Sq1 and Sq2) in mouse dynamin II gene for silencing its expression. The siRNA treatment was followed by a 48 hr incubation period prior to pDNA electrotransfection. Dynamin II and β-actin (loading control) expression levels in Western blot analysis are shown in Panel A and normalized electrotransfection efficiencies are shown in Panel B. The bars and error bars indicate the means and standard deviations of 4 independent trials, respectively. The data from each trial, used in mean and standard deviation calculation, was the average value of replicates or triplicates. *, P<0.05 (Mann-Whitney U test).

Mentions: B16-F10 cells were treated with either of two specific, small interfering RNA (siRNA) sequences directed against two different sequences in the mouse dynamin II gene (i.e., Sq1 and Sq2 described in the Materials and Methods section) or a negative control siRNA sequence with comparable GC content. Western blot analysis revealed that only siRNA sequence Sq1 resulted in sustained silencing of dynamin II expression (see Figure 5A) during the entire experimental period. The effects of dynamin II silencing on eTE of GFP-encoding pDNA are shown in Figure 5B. Cells treated with siRNA sequence Sq1 had significantly lower eTE than the control siRNA-treated cells (P<0.05). However, there was no statistically significant difference in eTE (P = 0.19) between cells treated with siRNA sequence Sq2 and the control siRNA. These data demonstrated that specific knockdown of dynamin II expression directly resulted in a ∼50% reduction in eTE.


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

Wu M, Yuan F - PLoS ONE (2011)

Effects of dynamin II knockdown on pDNA electrotransfection.B16-F10 cells were transfected with either the control siRNA or one of the two specific siRNA oligos directed against two different sequences (i.e., Sq1 and Sq2) in mouse dynamin II gene for silencing its expression. The siRNA treatment was followed by a 48 hr incubation period prior to pDNA electrotransfection. Dynamin II and β-actin (loading control) expression levels in Western blot analysis are shown in Panel A and normalized electrotransfection efficiencies are shown in Panel B. The bars and error bars indicate the means and standard deviations of 4 independent trials, respectively. The data from each trial, used in mean and standard deviation calculation, was the average value of replicates or triplicates. *, P<0.05 (Mann-Whitney U test).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113837&req=5

pone-0020923-g005: Effects of dynamin II knockdown on pDNA electrotransfection.B16-F10 cells were transfected with either the control siRNA or one of the two specific siRNA oligos directed against two different sequences (i.e., Sq1 and Sq2) in mouse dynamin II gene for silencing its expression. The siRNA treatment was followed by a 48 hr incubation period prior to pDNA electrotransfection. Dynamin II and β-actin (loading control) expression levels in Western blot analysis are shown in Panel A and normalized electrotransfection efficiencies are shown in Panel B. The bars and error bars indicate the means and standard deviations of 4 independent trials, respectively. The data from each trial, used in mean and standard deviation calculation, was the average value of replicates or triplicates. *, P<0.05 (Mann-Whitney U test).
Mentions: B16-F10 cells were treated with either of two specific, small interfering RNA (siRNA) sequences directed against two different sequences in the mouse dynamin II gene (i.e., Sq1 and Sq2 described in the Materials and Methods section) or a negative control siRNA sequence with comparable GC content. Western blot analysis revealed that only siRNA sequence Sq1 resulted in sustained silencing of dynamin II expression (see Figure 5A) during the entire experimental period. The effects of dynamin II silencing on eTE of GFP-encoding pDNA are shown in Figure 5B. Cells treated with siRNA sequence Sq1 had significantly lower eTE than the control siRNA-treated cells (P<0.05). However, there was no statistically significant difference in eTE (P = 0.19) between cells treated with siRNA sequence Sq2 and the control siRNA. These data demonstrated that specific knockdown of dynamin II expression directly resulted in a ∼50% reduction in eTE.

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