Preassembled Single-Stranded RNA-Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium
Preassembled Single-Stranded RNA-Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium
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DOI：
10.1093/infdis/jiv588
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发表时间：
2016-04-15
影响因子：
6.4
通讯作者：
White, A. Clinton, Jr.
中科院分区：
文献类型：
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作者：
Castellanos-Gonzalez, Alejandro;Perry, Nicolas;White, A. Clinton, Jr.
隐孢子虫病是全世界腹泻发病和死亡的常见原因。由于缺乏对隐孢子虫寄生虫进行基因操纵的方法，有关这种感染的研究进展已经放缓。小干扰 RNA (siRNA) 广泛用于研究基因功能，但隐孢子虫缺乏处理 siRNA 所需的酶。通过与人类酶 Argonaute 2 (hAgo2) 和隐孢子虫单链 RNA (ssRNA) 预组装复合物，我们诱导了隐孢子虫 RNA 靶标的特异性切片。我们通过用 ssRNA-hAgo2 复合物转染活寄生虫，证明了 mRNA 和蛋白质水平上靶基因表达的减少。此外，我们使用这种方法来确认所选分子在宿主细胞侵袭过程中的作用。这种新方法提供了一种沉默隐孢子虫基因的新方法，以研究它们在宿主-寄生虫相互作用中的作用以及作为化疗的潜在靶标。
Cryptosporidiosis is a common cause of diarrhea morbidity and mortality worldwide. Research progress on this infection has been slowed by lack of methods to genetically manipulate Cryptosporidium parasites. Small interfering RNA (siRNA) is widely used to study gene function, but Cryptosporidium species lack the enzymes necessary to process siRNA. By preassembling complexes with the human enzyme Argonaute 2 (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA), we induced specific slicing in Cryptosporidium RNA targets. We demonstrated the reduction in expression of target genes at the mRNA and protein levels by transfecting live parasites with ssRNA-hAgo2 complexes. Furthermore we used this method to confirm the role of selected molecules during host cell invasion. This novel method provides a novel means of silencing Cryptosporidium genes to study their role in host-parasite interactions and as potential targets for chemotherapy.