Enhanced gene transfection using calcium phosphate co-precipitates and low-intensity pulsed ultrasound
Enhanced gene transfection using calcium phosphate co-precipitates and low-intensity pulsed ultrasound
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DOI：
10.1016/j.ejps.2012.08.007
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发表时间：
2012-11-20
影响因子：
4.6
通讯作者：
Kondo, Takashi
中科院分区：
文献类型：
--
作者：
Hassan, Mariame A.;Ahmed, Iman S.;Kondo, Takashi
通过超声波(US)可控地破坏细胞膜，从而促进外源物种进入的能力，现在已经达到了一定的成熟状态。然而，一个令人信服的问题是，美国在促进转基因方面是否还有剩余的作用：即，一旦遗传物质被输送到细胞质，美国能否帮助其进入细胞核？本实验就是为了解决这个问题而设计的。因此，我们的实验装置区分：(I)前体细胞膜渗透步骤，和(Ii)任何随后的细胞内转运进入细胞核。在这项研究中，磷酸钙共沉淀物(CAP)被用来内化HeLa细胞中编码荧光素酶的质粒DNA(PDNA-Luc)(>；90%)。与CaP-PDNA-Luc共孵育2小时后，洗涤和辐照细胞不同时间。结果表明，当实施更长的照射时间时，US确实可以增强先前内化的基因在细胞内的运输，最终导致从增强的荧光素酶表达推断的成功核定位的可能性增加。此外，结果表明，US在细胞内的作用可能是通过一种似乎不限于破坏内体小泡稳定的途径来调节的。因此，这项研究提供了有关US的细胞内效应的新信息，实际上代表了一种将US和CAP载体相结合以提高基因传递效率的新方式。(C)2012爱思唯尔B.V.保留所有权利。
The capability to controllably disrupt the cell membrane by ultrasound (US), thus facilitating entry of exogenous species, has now reached a state of some maturity. However, a compelling question asks whether there is a residual role for US in enhancing transfection: that is, once the genetic material has been delivered to the cytosol, can US assist in its transport into the nucleus? The present experiment was designed with a view to addressing this question. As such, our experimental setup discriminates between: (i) the precursor cell membrane permealization step, and (ii) any subsequent intracellular trafficking into the nucleus. In this study, calcium phosphate co-precipitates (CaP) were used to internalize plasmid DNA encoding for luciferase (pDNA-Luc) (>90%) in HeLa cells. After 2 h incubation with the CaP-pDNA-Luc, cells were washed and insonated for varying durations. The results showed that US can indeed enhance the intracellular trafficking of previously internalized genes when longer insonation periods are implemented, culminating with an increased probability for successful nuclear localization, as inferred from an enhanced luciferase expression. Moreover, the results suggest that the intracellular role of US might be mediated through a pathway that appears not to be limited to destabilizing the endosomal vesicles. The study thus provides new information regarding the intracellular effects of US, and in effect represents a new modality combining US and CaP carriers for improved efficiency in gene delivery. (C) 2012 Elsevier B.V. All rights reserved.