Protocols for gene silencing in schistosomes
Protocols for gene silencing in schistosomes
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DOI：
10.1016/j.exppara.2007.07.012
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发表时间：
2007-11-01
影响因子：
2.1
通讯作者：
Skelly, Patrick J.
中科院分区：
文献类型：
--
作者：
Ndegwa, David;Krautz-Peterson, Greice;Skelly, Patrick J.
血吸虫是一种寄生的扁形蠕虫，全球有超过2亿人感染。近年来，在血吸虫基因组学和蛋白质组学以及用于这些寄生虫的分子工具的开发方面取得了许多进展。其中更有前景的方法是RNA干扰(RNAi)，这是一种基因特异性双链RNA(DsRNA)引发同源mRNA转录本降解的机制。我们的目标是开发利用RNAi的有效方案，用于曼氏血吸虫在哺乳动物内的生活阶段。在这项工作中，编码碱性磷酸酶(SMAP)的基因是通过将寄生虫暴露于编码SMAP编码区的dsRNA来定位的。已知SMAP在多种寄生虫组织中表达。我们报道了长dsRNAs和合成的短抑制性RNAs(SiRNAs)都能有效地在培养的血吸虫幼虫和成年雄性和雌性血吸虫中诱导SMAP基因抑制。电穿孔作为dsRNA传递的一种方式比简单地将寄生虫浸泡在同等剂量下更有效。用实时定量聚合酶链式反应检测治疗2天后的相对SMAP RNA水平，比对照组低90%。与SMAP RNA的这种下降相称的是，在治疗后5天，检测到的相对碱性磷酸酶活性水平比对照组低70%。这里描述的方案导致血管内血吸虫靶基因的强烈抑制，可能具有广泛的适用性，并促进血吸虫功能基因组学。(C)2007 Elsevier Inc.保留所有权利。
Schistosomes are parasitic platyhelminths that infect over 200 million people globally. In recent years there have been many advances in schistosome genomics and proteomics and in the development of molecular tools for use with these parasites. Among the more promising methodologies is RNA interference (RNAi) which is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. We aim to develop effective protocols utilizing RNAi for use in the intra-mammalian life stages of Schistosoma mansoni. In this work, the gene encoding alkaline phosphatase (SmAP) was targeted by exposing the parasites to dsRNA encoding part of the SmAP coding region. SmAP is known to be expressed in a variety of parasite tissues. We report that both long dsRNAs as well as synthetic short inhibitory RNAs (siRNAs) are effective at eliciting SmAP gene suppression in cultured schistosomula and in adult males and females. Electroporation as a mode of dsRNA delivery is more efficient than simply soaking the parasites in an equivalent dose. Relative SmAP RNA levels >90% lower than controls were routinely detected, when measured 2 days after treatment by electroporation, using quantitative real-time PCR. Commensurate with this decline in SmAP RNA, relative alkaline phosphatase enzyme activity levels >70% lower than controls were detected, 5 days after treatment. Protocols described here that result in the robust suppression of target genes in intravascular schistosomes may have wide applicability and promote functional schistosome genomics. (C) 2007 Elsevier Inc. All rights reserved.