Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells
Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells
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DOI：
10.1016/j.ibmb.2013.05.001
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发表时间：
2013-08-01
影响因子：
3.8
通讯作者：
Swevers, Luc
中科院分区：
文献类型：
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作者：
Kolliopoulou, Anna;Swevers, Luc
尽管鳞翅目细胞培养物在转染实验中显示出对RNAi的有效响应，但鳞翅目昆虫体内dsRNA介导的基因沉默实验的特征在于高变异性。为了鉴定调节鳞翅目昆虫RNAi反应的核心RNAi因子，我们采用蚕蛾卵巢衍生的Bm5细胞作为测试系统，因为已知该细胞系在dsRNA转染后在沉默中有效地响应。使用了两种平行的方法;涉及敲低核心RNAi基因或过表达主要siRNA途径因子，以分别研究RNAi沉默应答的可能抑制或刺激。发现来自所有三种主要小RNA途径的组分（miRNA的BmAgo-1、siRNA的BmAgo-2/BmDcr-2和piRNA的BmAgo-3）参与由dsRNA触发的RNAi应答。由于BmAgo-3（一种在果蝇中独立于Dicer发挥功能的piRNA途径中的因子）被鉴定为RNAi应答中的限制因子，因此还测试了正义和反义ssRNA以诱导基因沉默，但证明是无效的，这表明BmAgo-3在家蚕中的dsRNA依赖性作用。在主要siRNA因子的有效过表达后，免疫荧光染色显示Bm5细胞中的主要细胞质定位。这是在鳞翅目昆虫中的首次研究，为使用转染的B调节dsRNA介导的沉默反应中所有三种已知的小RNA途径的可能重叠提供了证据。Mori来源的Bm5细胞作为实验系统。(C)2013爱思唯尔有限公司保留所有权利。
Experiments of dsRNA-mediated gene silencing in lepidopteran insects in vivo are characterized by high variability although lepidopteran cell cultures have shown an efficient response to RNAi in transfection experiments. In order to identify the core RNAi factors that regulate the RNAi response of Lepidoptera, we employed the silkmoth ovary-derived Bm5 cells as a test system since this cell line is known to respond potently in silencing after dsRNA transfection. Two parallel approaches were used; involving knockdown of the core RNAi genes or over-expression of the main siRNA pathway factors, in order to study possible inhibition or stimulation of the RNAi silencing response, respectively. Components from all three main small RNA pathways (BmAgo-1 for miRNA, BmAgo-2/BmDcr-2 for siRNA, and BmAgo-3 for piRNA) were found to be involved in the RNAi response that is triggered by dsRNA. Since BmAgo-3, a factor in the piRNA pathway that functions independent of Dicer in Drosophila, was identified as a limiting factor in the RNAi response, sense and antisense ssRNA was also tested to induce gene silencing but proved to be ineffective, suggesting a dsRNA-dependent role for BmAgo-3 in Bombyx mori. After efficient overexpression of the main siRNA factors, immunofluorescence staining revealed a predominant cytoplasmic localization in Bm5 cells. This is the first study in Lepidoptera to provide evidence for possible overlapping of all three known small RNA pathways in the regulation of the dsRNA-mediated silencing response using transfected B. mori-derived Bm5 cells as experimental system. (C) 2013 Elsevier Ltd. All rights reserved.