Rosa Bruce A, McNulty Samantha N, Mitreva Makedonka, Jasmer Douglas P
The McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
The McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA; Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Genetics, Washington University School of Medicine, St. Louis, MO 63108, USA.
Int J Parasitol. 2017 Apr;47(5):271-279. doi: 10.1016/j.ijpara.2016.12.005. Epub 2017 Feb 20.
Ascaris suum provides a powerful model for studying parasitic nematodes, including individual tissues such as the intestine, an established target for anthelmintic treatments. Here, we add a valuable experimental component to our existing functional, proteomic, transcriptomic and phylogenomic studies of the Ascaris suum intestine, by developing a method to manipulate intestinal cell functions via direct delivery of experimental treatments (in this case, double-stranded (ds)RNA) to the apical intestinal membrane. We developed an intestinal perfusion method for direct, controlled delivery of dsRNA/heterogeneous small interfering (hsi) RNA into the intestinal lumen for experimentation. RNA-Seq (22 samples) was used to assess influences of the method on global intestinal gene expression. Successful mRNA-specific knockdown in intestinal cells of adult A. suum was accomplished with this new experimental method. Global transcriptional profiling confirmed that targeted transcripts were knocked down more significantly than any others, with only 12 (0.07% of all genes) or 238 (1.3%) off-target gene transcripts consistently differentially regulated by dsRNA treatment or the perfusion experimental design, respectively (after 24h). The system supports controlled, effective delivery of treatments (dsRNA/hsiRNA) to the apical intestinal membrane with relatively minor off-target effects, and builds on our experimental model to dissect A. suum intestinal cell functions with broad relevance to parasitic nematodes.
猪蛔虫为研究寄生线虫提供了一个强大的模型，包括如肠道等单个组织，而肠道是驱虫治疗的既定靶点。在此，我们通过开发一种方法，即通过将实验处理（在本研究中为双链（ds）RNA）直接递送至肠道顶端膜来操纵肠道细胞功能，为我们现有的猪蛔虫肠道功能、蛋白质组学、转录组学和系统基因组学研究增添了一个有价值的实验组成部分。我们开发了一种肠道灌注方法，用于将dsRNA/异质小干扰（hsi）RNA直接、可控地递送至肠腔进行实验。RNA测序（22个样本）用于评估该方法对整体肠道基因表达的影响。利用这种新的实验方法成功实现了成年猪蛔虫肠道细胞中特定mRNA的敲低。整体转录谱分析证实，靶向转录本的敲低比其他任何转录本都更显著，在dsRNA处理或灌注实验设计后（24小时后），分别只有12个（占所有基因的0.07%）或238个（1.3%）脱靶基因转录本持续受到差异调节。该系统支持将处理（dsRNA/hsiRNA）可控、有效地递送至肠道顶端膜，且脱靶效应相对较小，并基于我们的实验模型来剖析与寄生线虫广泛相关的猪蛔虫肠道细胞功能。
