Knockdown of chimeric glucocerebrosidase by green fluorescent protein-directed small interfering RNA.
Knockdown of chimeric glucocerebrosidase by green fluorescent protein-directed small interfering RNA.
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通过绿色荧光蛋白引导的小干扰 RNA 敲低嵌合葡萄糖脑苷脂酶。
DOI：
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发表时间：
2004
影响因子：
0.4
通讯作者：
F. Choy
中科院分区：
文献类型：
--
作者：
T. Campbell;F. Choy
高谢病是最常见的溶酶体储存障碍类型，其特征是膜相关水解酶--葡萄糖脑苷酶的遗传性缺陷。葡萄糖脑苷酶催化葡萄糖脑苷水解成神经酰胺和葡萄糖，这是膜鞘糖脂循环的关键步骤。目前高谢病的治疗标准--酶替代疗法费用过高，使许多人无法接受治疗。这一局限性导致了对更有效和更具成本效益的蛋白质生产方法和替代疗法的广泛探索，导致对葡萄糖脑苷酶的生物合成和当前治疗技术的更密切的研究。使用特定的小干扰RNA(SiRNAs)来敲除靶基因是研究这一过程的一个有吸引力的选择，尽管尚未有针对葡萄糖脑苷酶的siRNA的报道。然而，我们注意到，绿色荧光蛋白(GFP)导向的siRNAs不仅可以提供一个阳性对照来测试siRNA的传递和系统完整性，而且还可以作为一种手段来击倒融合伙伴，而不必设计针对该伙伴的siRNAs。在有效地将增强型绿色荧光蛋白(EGFP)标记的葡萄糖脑苷酶构建物和GFP导向的siRNAs共转染COS-1细胞后，我们报告了在RNA和蛋白质水平上成功地击倒了所有含有EGFP的构建物。这提供了一种检查酶生物合成和处理选项的方法。此外，这项技术也适用于其他系统，因为我们已经证明了当GFP与另一个感兴趣的基因融合时，在哺乳动物细胞中作为siRNA靶标的有效性。
Gaucher disease, the most common type of lysosomal storage disorder, is characterized by an inherited deficiency of the membrane-associated hydrolase, glucocerebrosidase. Glucocerebrosidase catalyzes the hydrolysis of glucocerebroside to ceramide and glucose, a crucial step in the recycling of membrane sphingolipids. The exorbitant cost of the current treatment standard for Gaucher disease, enzyme replacement therapy, prevents many from receiving treatment. This limitation has led to a wide-spread search for more efficient and cost-effective methods of protein production and alternate therapies, resulting in a closer examination of glucocerebrosidase biosynthesis and current treatment techniques. The use of specific small interfering RNAs (siRNAs) to knock down target genes is an attractive option for studying such processes, though a glucocerebrosidase-specific siRNA has yet to be reported. We note, however, that green fluorescent protein (GFP)-directed siRNAs can not only provide a positive control to test siRNA delivery and system integrity, but also serve as a means to knock down a fusion partner without having to design siRNAs specific to the partner. After effectively co-transfecting COS-1 cells with enhanced GFP (EGFP)-tagged glucocerebrosidase constructs and GFP-directed siRNAs, we report successful knockdown of all EGFP-containing constructs at both the RNA and protein levels. This provides a method of examining enzyme biosynthesis and treatment options. Furthermore, this technique is applicable to other systems, since we have demonstrated the usefulness of GFP as a siRNA target in mammalian cells when fused to another gene of interest.
影响因子：
11.4
作者：
Shi, Y
通讯作者：
Shi, Y
人葡萄糖脑苷脂酶基因具有两个功能性 ATG 起始密码子。
影响因子：
9.8
作者：
Sorge,JA;West,C;Kuhl,W;Treger,L;Beutler,E
通讯作者：
Beutler,E