Kocan Katherine M, Blouin Edmour, de la Fuente José
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, OK, USA.
J Vis Exp. 2011 Jan 20(47):2474. doi: 10.3791/2474.
Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases(1) and the most important vectors affecting cattle industry worldwide(2). Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions(3). Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health(4,5). RNA interference (RNAi)(6) is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger(7-9). Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications(10). RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable(5,11). The genetic characterization of ticks has been limited until the recent application of RNAi(12,13). In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens(14). Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens(15-19).
蜱是野生和家养动物以及人类的专性吸血外寄生虫，被认为是全球仅次于蚊子的人类疾病传播媒介(1)，也是影响全球养牛业的最重要传播媒介(2)。蜱属于蜱螨亚纲、寄螨目、硬蜱亚目，分布于从北极到热带的世界各地(3)。尽管人们努力控制蜱虫侵扰，但这些外寄生虫仍然是人类和动物健康的严重问题(4,5)。RNA干扰（RNAi）(6)是一种基于核酸的反向遗传学方法，涉及破坏基因表达以确定基因功能或其对代谢途径的影响。小干扰RNA（siRNA）是RNAi途径的效应分子，该途径由双链RNA（dsRNA）启动，导致含有与dsRNA触发物相同序列的细胞质mRNA发生有效的序列特异性降解(7-9)。迄今为止，在所有研究过的真核生物中都发现了由dsRNA引发的转录后基因沉默机制，并且RNAi已在多种生物体中迅速发展成为功能基因组学研究和其他应用的工具(10)。在蜱以及其他无法获得或不可靠的遗传操作替代方法的生物体中，RNAi已成为使用最广泛的基因沉默技术(5,11)。在RNAi最近应用之前，蜱的遗传特征一直有限(12,13)。在RNAi可用的短时间内，它已被证明是研究蜱基因功能、蜱-病原体界面特征以及蜱保护性抗原筛选和特征分析的有价值工具(14)。本文描述了一种通过向未进食的蜱虫注射dsRNA进行RNA干扰的方法。从这种实验方法中获得的知识可能会显著有助于理解基本生物系统以及开发控制蜱虫侵扰和预防蜱传病原体传播的疫苗(15-19)。
