Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts
Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts
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DOI：
10.1016/s0166-6851(03)00078-1
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发表时间：
2003-05-01
影响因子：
1.5
通讯作者：
Yoshino, TP
中科院分区：
文献类型：
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作者：
Boyle, JP;Wu, XJ;Yoshino, TP
缺乏改变基因表达的有效工具，对寄生虫中基因功能的直接评估受到了阻碍。本研究的目的是使用RNA脱粒（RNAI）在人类血液氟曼森菌的幼虫阶段实现靶向基因敲低。我们选择了两个用于RNAi实验的链球菌基因：SGTP1，一种促进扩散葡萄糖转运蛋白和3-磷酸甘油醛脱氢酶（GAPDH）。当通过特有的两个基因之一的dsRNA在体外处理6天的曼氏菌幼虫时，通过定量PCR（qPCR）确定，靶向转录水平减少了70-80％，而未靶向的转录本不受影响。寄生虫暴露于SGTP1 dsRNA，但不暴露于GAPDH DSRNA，将幼虫葡萄糖摄取能力降低了40％，这表明SGTP1转录本敲低导致葡萄糖转运活性减少的功能表型。初始DSRNA治疗后长达28天，DSRNA处理对转录水平的影响很明显。有趣的是，只有在允许Miracidia在存在下向孢子囊过渡到孢子囊的情况下，DSRNA处理才有效，而完全转化的孢子囊肿的治疗无效。暴露于若丹明标记的dsRNA的幼虫中的荧光模式是奇迹和孢子细胞相似，这表明两个生命阶段之间对DSRNA处理的易感性差异可能不是由于进入DSRNA的差异。总体而言，这项技术将能够直接评估单个基因在曼森幼虫阶段的生理过程中的作用，这是确定这种重要人类病原体的新干预靶标的至关重要的一步。 （c）2003 Elsevier Science B.V.保留所有权利。
Direct assessments of gene function in parasitic flatworms have been hampered by the lack of effective tools to alter gene expression. The aim of the present study was to use RNA-interference (RNAi) to achieve targeted gene knockdown in larval stages of the human blood fluke, Schistosoma mansoni. We selected two S. mansoni genes for RNAi experiments: SGTP1, a facilitated diffusion glucose transporter and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When S. mansoni larvae were treated in vitro for 6 days with dsRNA specific to one of these two genes, targeted transcript levels were reduced by 70-80% as determined by quantitative PCR (qPCR), while non-targeted transcripts were unaffected. Parasite exposure to SGTP1 dsRNA, but not GAPDH dsRNA, reduced larval glucose-uptake capacity by 40%, demonstrating that SGTP1 transcript knockdown results in the functional phenotype of reduced glucose transport activity. The effect of dsRNA treatment on transcript level was evident for up to 28 days after an initial dsRNA treatment. Interestingly, dsRNA treatment was effective only when miracidia were allowed to undergo the transition to sporocysts in its presence, while treatment of fully transformed sporocysts was ineffective. Fluorescence patterns in larvae exposed to rhodamine-labeled dsRNA as miracidia and sporocysts were similar, suggesting that the difference in susceptibility to dsRNA treatment between the two life stages may not be due to differences in dsRNA entry. Overall, this technology will enable direct assessment of the roles of individual genes in physiological processes of larval stages of S. mansoni, a crucial step in the identification of novel intervention targets for this important human pathogen. (C) 2003 Elsevier Science B.V. All rights reserved.