Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis.
Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis.
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慢病毒转导促进了线虫寄生虫nippostrongylus brasiliensis中的RNA干扰。
DOI：
10.1371/journal.ppat.1009286
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发表时间：
2021-01
期刊：
影响因子：
6.7
通讯作者：
Selkirk ME
中科院分区：
文献类型：
--
作者：
Hagen J;Sarkies P;Selkirk ME
迄今为止，动物寄生线虫在很大程度上难以进行遗传操作，并且在大多数情况下，用于影响RNA干扰（RNAi）的方法无效或不一致。我们在这里描述了一种新的方法来遗传操作的Nippostrongylus brasiliensis，胃肠线虫感染的一个广泛使用的实验室模型。成功地用水疱性口炎病毒糖蛋白G （VSV-G）假型慢病毒转导了巴西乳螨。该病毒经线虫肠道吸收，RNA逆转录为原病毒DNA，在感染幼虫中稳定产生转基因转录物，导致报告蛋白mCherry的表达。通过将秀丽隐杆线虫的hlh11启动子和tbb2 3´-UTR结合到病毒构建体中，提高了转基因表达。以这种方式传递的适应microrna的短发夹rna被正确处理，导致β-微管蛋白同型1 （tbb-iso-1）的部分敲低和分泌乙酰胆碱酯酶B （ache-B）。该系统通过慢病毒递送双链rna进一步完善，双链rna在22g - rna的加工和产生后作为RNAi的触发器。在F1卵和第三期幼虫中检测到病毒编码序列，表明前病毒DNA进入种系并具有遗传性。因此，慢病毒转导为寄生线虫的基因操作提供了一种新的手段，包括基因沉默和外源基因的表达。寄生线虫复杂的生命周期使得对它们进行遗传操作非常困难，而在其他生物体中常用的删除或沉默基因的方法对大多数感染动物的线虫是无效的。这阻碍了了解特定基因和蛋白质的功能，以及它们在线虫寄生虫与宿主的发育和相互作用中的作用。我们在这里展示了外源遗传物质可以通过使用病毒载体引入广泛使用的肠道线虫感染实验室模型。对该载体进行修饰以提高转基因表达，并通过转导的线虫幼虫在体外表达一个报告蛋白。随后，我们利用病毒载体将双链RNA分子传递给幼虫。这些分子沿着已知的途径加工，导致两个测试基因的部分敲低。该系统代表了一种遗传操纵线虫寄生虫的新手段，除了定义控制感染的新靶点外，还将有助于了解其复杂的生物学。
Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of β-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes. The complex life cycle of parasitic nematodes makes them very difficult to manipulate genetically, and methods to delete or silence genes which are routinely used in other organisms are ineffective in most species of nematodes which infect animals. This has hindered attempts to understand the function of defined genes and proteins, and their roles in development and interaction of nematode parasites with their host. We show here that foreign genetic material can be introduced into a widely used laboratory model of intestinal nematode infection by using a viral vector. The vector was modified to improve transgene expression, and a reporter protein expressed by transduced nematode larvae in vitro. We subsequently utilised the viral vector to deliver double stranded RNA molecules to the larvae. These molecules were processed along known pathways, resulting in partial knockdown of two test genes. This system represents a new means of genetically manipulating nematode parasites, and will aid in understanding their complex biology, in addition to defining new targets for control of infection.
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