Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level
Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level
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DOI：
10.1002/jgm.2706
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发表时间：
2013-05-01
影响因子：
3.5
通讯作者：
Pavlin, Mojca
中科院分区：
文献类型：
--
作者：
Haberl, Sasa;Kanduser, Masa;Pavlin, Mojca
基因电转移是一种用于将DNA传递到细胞中的非病毒方法。这涉及到几个步骤。其中之一是DNA与细胞膜的相互作用，这在DNA进入细胞之前是至关重要的。我们分析了DNA膜相互作用的水平与电转移效率和DNA在细胞膜上的电泳积累的重要性。系统比较了长持续时间、短持续时间以及电渗透短脉冲（高压）和电泳长脉冲（低压）的组合。探讨了Mg2+离子浓度对电转移的影响及其对dna酶活性的影响。方法利用TOTO-1标记的DNA可视化DNA膜相互作用。用编码绿色荧光蛋白的质粒DNA评价转染效率。结果使用较长脉冲可获得较高的相对电传递效率，而使用较短脉冲可保持细胞活力。与长时间脉冲（12%）相比，短时间脉冲使总转染率更高（24%），尽管观察到更高的dnama膜相互作用。在不同的HV-LV脉冲方案之间转染没有显著差异，尽管在HV+LV脉冲中观察到最高的dname膜相互作用。dna膜复合物的形成依赖于Mg2+浓度，而dna酶抑制剂不影响基因表达。结论基因电转移是一个复杂的过程，许多因素相互影响，dna膜的相互作用只是第一步。我们发现，较长的电脉冲可以获得较高的转染效率，但会降低存活能力，而较短的电脉冲可以获得中等的转染效率并保持存活能力。因此，每个应用程序都需要仔细选择脉冲协议。版权所有：John Wiley & Sons， Ltd。
Background Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNAmembrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg2+ ion concentrations on electrotransfer and their effect on DNase activity were explored. Methods To visualize the DNAmembrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein. Results Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to long-duration pulses (12%), although a higher DNAmembrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNAmembrane interaction was observed with HV+LV pulses. The formation of the DNAmembrane complex depended on the Mg2+ concentration, whereas DNase inhibitor did not affect gene expression. Conclusions Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNAmembrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol. Copyright (c) 2013 John Wiley & Sons, Ltd.