High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking, and function.
High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking, and function.
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DOI：
10.1186/1750-1326-5-17
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发表时间：
2010-04-21
影响因子：
15.1
通讯作者：
Rossoll W
中科院分区：
文献类型：
--
作者：
Fallini C;Bassell GJ;Rossoll W
培养的脊髓运动神经元是研究发育、轴突生长和寻路的基本机制的有价值的工具，重要的是，可以分析运动神经元疾病的病理机制。然而，这种细胞培养模型的应用受到缺乏可用于其他神经元的有效基因转移技术的限制。为了解决这个问题，我们已经建立了磁转染作为一种新的方法，简单而有效地转染小鼠胚胎运动神经元。该技术允许研究基因表达和沉默对运动神经元发育和存活的影响。我们发现，磁转染，一种新的转染技术的基础上交付的DNA包被的磁性纳米珠，可用于转染初级运动神经元。因此，为了使用这种方法作为研究轴突蛋白定位和转运的新工具，我们优化了条件并确定了有效转染率>45%的参数，同时最大限度地减少了对存活和形态的毒性影响。为了证明这种方法的潜力，我们已经使用了转染与质粒编码的荧光融合蛋白显示的脊髓性肌萎缩症蛋白Smn是积极运输沿着轴突的活的初级运动神经元，支持一个轴突特异性的作用Smn是不同的，从其典型的功能在mRNA剪接。我们还能够通过显着降低细胞体和轴突中的Smn水平来显示磁转染对基于shRNA的构建体的基因敲低的适用性，为研究运动神经元中轴突蛋白的功能开辟了新的机会。在这项研究中，我们已经建立了一个优化的磁转染方案作为一种新的转染方法，为原代运动神经元，是简单，高效和无毒的。我们预计，这种新的方法将有一个广泛的适用性在运动神经元发育，轴突运输，运动神经元疾病的分子机制的研究。
Cultured spinal motor neurons are a valuable tool to study basic mechanisms of development, axon growth and pathfinding, and, importantly, to analyze the pathomechanisms underlying motor neuron diseases. However, the application of this cell culture model is limited by the lack of efficient gene transfer techniques which are available for other neurons. To address this problem, we have established magnetofection as a novel method for the simple and efficient transfection of mouse embryonic motor neurons. This technique allows for the study of the effects of gene expression and silencing on the development and survival of motor neurons. We found that magnetofection, a novel transfection technology based on the delivery of DNA-coated magnetic nanobeads, can be used to transfect primary motor neurons. Therefore, in order to use this method as a new tool for studying the localization and transport of axonal proteins, we optimized conditions and determined parameters for efficient transfection rates of >45% while minimizing toxic effects on survival and morphology. To demonstrate the potential of this method, we have used transfection with plasmids encoding fluorescent fusion-proteins to show for the first time that the spinal muscular atrophy-disease protein Smn is actively transported along axons of live primary motor neurons, supporting an axon-specific role for Smn that is different from its canonical function in mRNA splicing. We were also able to show the suitability of magnetofection for gene knockdown with shRNA-based constructs by significantly reducing Smn levels in both cell bodies and axons, opening new opportunities for the study of the function of axonal proteins in motor neurons. In this study we have established an optimized magnetofection protocol as a novel transfection method for primary motor neurons that is simple, efficient and non-toxic. We anticipate that this novel approach will have a broad applicability in the study of motor neuron development, axonal trafficking, and molecular mechanisms of motor neuron diseases.
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影响因子：
4.8
作者：
Dalby, B;Cates, S;Ciccarone, VC
通讯作者：
Ciccarone, VC
影响因子：
48
作者：
Riedl, Julia;Crevenna, Alvaro H.;Wedlich-Soldner, Roland
通讯作者：
Wedlich-Soldner, Roland
影响因子：
4
作者：
Hyun, Chang;Young, Eun;Park, Se Pill
通讯作者：
Park, Se Pill
影响因子：
2.7
作者：
Wirth, Brunhilde;Brichta, Lars;Hahnen, Eric
通讯作者：
Hahnen, Eric
影响因子：
4.7
作者：
Cisterni, C;Henderson, CE;Déglon, N
通讯作者：
Déglon, N