RNAi screening in Drosophila cells identifies new modifiers of mutant huntingtin aggregation.
RNAi screening in Drosophila cells identifies new modifiers of mutant huntingtin aggregation.
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DOI：
10.1371/journal.pone.0007275
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发表时间：
2009-09-30
期刊：
影响因子：
3.7
通讯作者：
Nukina N
中科院分区：
文献类型：
--
作者：
Doumanis J;Wada K;Kino Y;Moore AW;Nukina N
黑腹果蝇是研究人类神经退行性疾病的理想模型。利用RNAi，我们已经进行了高通量筛选的果蝇幼虫CNS衍生细胞表达突变的人亨廷顿蛋白外显子1融合到EGFP与扩展的多聚谷氨酰胺重复序列（62 Q）的聚集体形成的修饰剂。7200个基因，涵盖了果蝇基因组的50%左右，进行了筛选，导致404个候选人的鉴定，无论是抑制或增强聚集。这些候选物在正常长度（18 Q）表达细胞中进行二次筛选，并修剪以去除具有大于10个脱靶效应（OTE）的dsRNA。设计并合成了剩余68个候选者的从头RNAi探针。在第三轮筛选后，在体内分析了21种高置信度候选物修饰突变亨廷顿蛋白诱导的眼变性和脑聚集的能力。我们已经建立了有用的模型，用于研究人类HD使用苍蝇，并通过我们的RNAi屏幕，我们已经确定了新的修饰剂的突变人类亨廷顿蛋白聚集和聚集体形成的大脑。新发现的修饰基因包括与核转运、核苷酸加工和信号传导相关的基因，可能参与多聚谷氨酰胺聚集体形成和亨廷顿病级联反应。
The fruitfly Drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. Utilizing RNAi, we have carried out a high-throughput screen for modifiers of aggregate formation in Drosophila larval CNS-derived cells expressing mutant human Huntingtin exon 1 fused to EGFP with an expanded polyglutamine repeat (62Q). 7200 genes, encompassing around 50% of the Drosophila genome, were screened, resulting in the identification of 404 candidates that either suppress or enhance aggregation. These candidates were subjected to secondary screening in normal length (18Q)-expressing cells and pruned to remove dsRNAs with greater than 10 off-target effects (OTEs). De novo RNAi probes were designed and synthesized for the remaining 68 candidates. Following a tertiary round of screening, 21 high confidence candidates were analyzed in vivo for their ability to modify mutant Huntingtin-induced eye degeneration and brain aggregation. We have established useful models for the study of human HD using the fly, and through our RNAi screen, we have identified new modifiers of mutant human Huntingtin aggregation and aggregate formation in the brain. Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades.
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影响因子：
7.8
作者：
Yamamoto, Ai;Cremona, M Laura;Rothman, James E
通讯作者：
Rothman, James E
DOI：
10.1073/pnas.110149597
发表时间：
2000-06-06
影响因子：
11.1
作者：
Clemens, JC;Worby, CA;Dixon, JE
通讯作者：
Dixon, JE
影响因子：
48
作者：
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通讯作者：
Mathey-Prevot, Bernard
影响因子：
30.8
作者：
Ravikumar, B;Vacher, C;Rubinsztein, DC
通讯作者：
Rubinsztein, DC
影响因子：
14.9
作者：
Arziman Z;Horn T;Boutros M
通讯作者：
Boutros M