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.
复制标题
DOI：
10.1371/journal.pone.0020923
复制
发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Yuan F
中科院分区：
文献类型：
--
作者：
Wu M;Yuan F
电场介导的基因传递或电转染是一种广泛应用于从基础细胞生物学研究到临床基因治疗的各种研究方法。然而，电转染法的机制仍存在争议。为此，我们研究了电转染效率(ETE)与质膜结合的依赖关系，以及三种内吞抑制剂(氯丙嗪、金雀异黄素、金雀异黄素)处理细胞或用特异性小干扰RNA(SiRNA)沉默Dynamin表达对ETE的影响。我们的数据表明，在电转染缓冲液中，二价阳离子(Ca~(2+)和Mg~(2+))的存在增强了PDNA在细胞膜上的吸附，因此，这种增强的吸附导致了ETE的增加，达到了每个阳离子的某个阈值浓度。经胰酶处理后10min，细胞膜上的pDNA被剥离，ETE显著减少，表明细胞完全摄取pDNA的时间(10~40min)远远超过了电场诱导的细胞膜瞬变孔的寿命(∼为10ms)。此外，用siRNA和所有三种药物抑制剂处理细胞后，ETE显著降低。这些发现表明，电转染依赖于两种机制：(I)pDNA与细胞膜的结合和(Ii)膜结合的pDNA的内吞作用。
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.
登录
查看更多内容
影响因子：
5
作者：
Haberl, Sasa;Miklavcic, Damijan;Pavlin, Mojca
通讯作者：
Pavlin, Mojca
影响因子：
3.5
作者：
Faurie, Cecile;Rebersek, Matej;Rols, Marie-Pierre
通讯作者：
Rols, Marie-Pierre
DOI：
10.1073/pnas.92.18.8328
发表时间：
1995-08-29
影响因子：
11.1
作者：
ARTALEJO, CR;HENLEY, JR;PALFREY, CH
通讯作者：
PALFREY, CH
DOI：
10.1073/pnas.0801763105
发表时间：
2008-08-19
影响因子：
11.1
作者：
Gratton, Stephanie E. A.;Ropp, Patricia A.;DeSimone, Joseph M.
通讯作者：
DeSimone, Joseph M.
影响因子：
3.4
作者：
DUGUID, J;BLOOMFIELD, VA;THOMAS, GJ
通讯作者：
THOMAS, GJ