Electrotransfer in differentiated myotubes: a novel, efficient procedure for functional gene transfer
Electrotransfer in differentiated myotubes: a novel, efficient procedure for functional gene transfer
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DOI：
10.1016/s0014-4827(03)00097-1
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发表时间：
2003-05-15
影响因子：
3.7
通讯作者：
Volpe, P
中科院分区：
文献类型：
--
作者：
Sandri, M;Bortoloso, E;Volpe, P
开发可靠的多核骨骼肌纤维基因表达实验操作技术对于理解生理学和病理生理学的分子机制至关重要。目前，病毒载体是在终末分化肌管中获得有效基因转移的唯一方法。在这里，我们提出了一种体外程序，该程序依赖于脉冲电场的应用，将裸露 DNA 转移到接种在盖玻片上的分化肌管中。通过荧光素酶含量的定量测定判断，与标准转染方法相比，电穿孔的效率至少高1000倍。转染肌管的百分比平均约为 45%。此外，我们成功地在肌管中转染了显性失活的ADP核糖基化因子1（ARF1）突变体，即ARF1N126I，从而干扰了内质网-高尔基体的运输，如顺式/内侧高尔基体标记GM130的亚细胞分布的改变所表明的。与β-半乳糖苷酶的共转染实验还表明，ARF1突变体似乎抑制成肌细胞融合，并且不能在肌管形成之前使用。目前的工作验证了电穿孔作为完全分化的肌管中基因转移的高效方法的使用。 (C) 2003 年爱思唯尔科学（美国）。版权所有。
Development of reliable techniques for experimental manipulation of gene expression in multinucleated skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and pathophysiology. At present, viral vectors represent the only method to obtain efficient gene transfer in terminally differentiated myotubes. Here we present an in vitro procedure that relies on the application of a pulsed electric field for transferring naked DNA into differentiated myotubes seeded on coverslips. Compared with standard transfection methods, electroporation was at least 1000 times more efficient, as judged by quantitative determination of luciferase content. Percentage of transfected myotubes averaged around 45%. Moreover, we were successful in transfecting a dominant-negative ADP ribosylation factor 1 (ARF1) mutant, i.e., ARF1N126I, in myotubes, thus interfering with endoplasmic reticulum-Golgi traffic, as indicated by alterations of subcellular distribution of GM130, a cis/medial-Golgi marker. Co-transfection experiments with beta-galactosidase also showed that the ARF1 mutant appeared to inhibit myoblast fusion and could not be used before myotube formation. The present work validates the use of electroporation as a highly efficient approach for gene transfer in fully differentiated myotubes. (C) 2003 Elsevier Science (USA). All rights reserved.