Tetracycline-regulated gene expression in the NSC-34-tTA cell line for investigation of motor neuron diseases
Tetracycline-regulated gene expression in the NSC-34-tTA cell line for investigation of motor neuron diseases
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DOI：
10.1016/j.molbrainres.2005.07.010
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发表时间：
2005-10-31
期刊：
影响因子：
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通讯作者：
Cantoni, L
中科院分区：
文献类型：
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作者：
Babetto, E;Mangolini, A;Cantoni, L
运动神经元样细胞系NSC-34已成为广泛用于运动神经元生物学和病理学研究的体外模型。我们通过稳定地转染编码四环素反式激活蛋白TTA的pTet-off，在该细胞系中建立了四环素调控的基因表达系统。用pBI-EGFP瞬时转染NSC-34-TTA细胞系，检测报告基因增强型绿色荧光蛋白的表达。我们用荧光显微镜和活细胞荧光定量分析评价了多西环素对ITA功能的调节。与NSC-34相比，调控最好的细胞株(NSC-34-tTA40)对报告基因荧光的诱导作用是NSC-34的66.4倍。蛋白质印迹和免疫细胞化学显示，α-微管蛋白、GAP-43和磷酸化的中丝和重神经丝在NSC-34-tTA40中有明显的表达，在NSC-34-tTA40和NSC-34中的表达相似。将该表达载体瞬时转入NSC-34-tTA40细胞，通过检测增强型绿色荧光蛋白和人铜锌超氧化物歧化酶的表达，验证了双向转录的功能。这两种蛋白都受多西环素的调节。这一新的细胞系NSC-34 tTA40允许四环素调控的基因表达，可能有助于揭开运动神经元变性的机制。(C)2005 Elsevier B.V.保留所有权利。
The motor neuron-like cell line NSC-34 has become a widely used in vitro model for motor neuron biology and pathology. We established a tetracycline-regulated gene expression system in this cell line by stably transfecting pTet-Off, which codifies for the tetracycline transactivator, the regulatory protein tTA. The monoclonal cell lines (NSC-34-tTA) were evaluated for the presence of functional tTA after transient transfection with pBI-EGFP, analyzing the expression of the reporter gene enhanced green fluorescent protein. We evaluated the regulation of ITA function with doxycycline using fluorescence microscopy and quantitative cytofluorimetric analysis on viable transfected cells. The best-regulated cell line (NSC-34-tTA40) had a 66.4-fold induction for the reporter gene fluorescence in comparison to NSC-34.Alpha-tubulin, GAP-43 and phosphorylated medium and heavy neurofilaments, proteins of importance for the motor neuronal phenotype, were evident in NSC-34-tTA40 by Western blot and immunocytochemistry; they were expressed similarly in NSC-34-tTA40 and in NSC-34.The cDNA of human Cu/Zn superoxide dismutase, a gene of interest for amyotrophic lateral sclerosis, was cloned into pBI-EGFP, downstream of the tetracycline-responsive bidirectional promoter. This plasmid was transiently transfected into NSC-34-tTA40, and the functionality of bidirectional transcription was verified by determining the expression of enhanced green fluorescent protein and of human Cu/Zn superoxide dismutase. Both proteins were regulated by doxycycline.This novel cell line, NSC-34 tTA40, that permits tetracycline-regulated gene expression may prove useful to unravel the mechanisms of motor neuron degeneration. (c) 2005 Elsevier B.V. All rights reserved.