In vitro gene manipulation of spinal muscular atrophy fibroblast cell line using gene-targeting fragment for restoration of SMN protein expression
In vitro gene manipulation of spinal muscular atrophy fibroblast cell line using gene-targeting fragment for restoration of SMN protein expression
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DOI：
10.1038/gt.2015.92
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发表时间：
2015-09
期刊：
影响因子：
5.1
通讯作者：
A. Rashnonejad;A. Rashnonejad;Cumhur Gündüz;S. Y. Susluer;H. Onay;B. Durmaz;M. Bandehpour;Ferda Ozkinay
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文献类型：
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作者：
A. Rashnonejad;A. Rashnonejad;Cumhur Gündüz;S. Y. Susluer;H. Onay;B. Durmaz;M. Bandehpour;Ferda Ozkinay
运动神经元（SMN）蛋白水平的降低，由SMN基因的纯合缺失引起，导致一种常见的神经退行性疾病，称为脊髓性肌萎缩症（SMA）。尽管广泛努力寻找SMA的治愈方法，但目前尚无有效治疗这种毁灭性疾病的方法。在这项研究中，试图通过“基因打靶”的方法在SMA成纤维细胞中恢复SMN的表达。我们设计了一个2697 bp的基因打靶盒，它由一个表达38 kD SMN蛋白的SMN 1开放阅读框和末端的SMN 1基因外显子1的上游和下游区域作为同源臂组成。使用Lipofectamine LTX-PLUS试剂通过基因靶向盒转染SMA成纤维细胞。通过PCR分析研究所选细胞中同源重组的发生。实时荧光定量PCR和免疫印迹分析显示SMN蛋白表达增加。免疫荧光分析结果表明，SMN核结构的数量，Gems，是相同的或大于在正常成纤维细胞中发现的Gems的数量。这项研究的结果表明，基因靶向方法，事实上，目前作为一种替代的SMA患者来源的细胞在体外恢复SMN的表达。
The reduced level of survival motor neuron (SMN) protein, caused by homozygous deletions in the SMN gene, led to a common neurodegenerative disorder known as spinal muscular atrophy (SMA). In spite of extensive efforts to find a cure for SMA, there is currently no effective treatment available for this devastating disease. In this study, restoration of SMN expression through ‘gene-targeting’method in SMA fibroblast cells was attempted. We designed a 2697-bp gene-targeting cassette; it consisted of an SMN1 open reading frame expressing 38 kD SMN protein and the upstream and downstream regions of exon 1 of SMN1 gene at the ends as the homology arms. SMA fibroblast cells were transfected by gene-targeting cassette using Lipofectamine LTX-PLUS reagent. Occurrence of homologous recombination in selected cells was investigated by PCR analysis. Increased expression of SMN protein was shown by real-time PCR and western blotting analysis. The immunofluorescence analysis results demonstrated that the number of SMN nuclear structures, Gems, was the same as or greater than the number of Gems found in normal fibroblasts. The results of this study indicate that gene-targeting methods do, in fact, present as an alternative for restoration of SMN expression in SMA patients-derived cells in vitro.