Verdonckt Thomas-Wolf, Vanden Broeck Jozef
Molecular Developmental Physiology and Signal Transduction Research Group, Animal Physiology and Neurobiology Division, Department of Biology, KU Leuven, Leuven, Belgium.
Front Physiol. 2022 Apr 13;13:836106. doi: 10.3389/fphys.2022.836106. eCollection 2022.
RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for experiments. In this paper, we describe and compare methods for the economical (43.2 €/mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the -specific gene and, as a non-endogenous control, the gene (). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates.
RNA干扰（RNAi）是一种高度保守的基因表达转录后调控途径。它已成为生命科学研究中的关键工具，在害虫管理应用方面具有广阔的潜力。为了诱导RNAi反应，必须将与靶基因特异的长双链RNA（dsRNA）序列递送至细胞。然后，这种dsRNA底物被加工成指导沉默反应的小RNA（sRNA）片段。应用该技术的一个主要障碍是需要以非常经济高效的方式生产足够大量的dsRNA。为了克服这个问题，人们对生物生产系统的开发和优化给予了很多关注。一种这样的系统是用L4440载体转化的HT115菌株。虽然已经证明其通过喂食细菌在动物中诱导基因敲低的有效性，但关于细菌衍生的dsRNA在实验中的适用性的知识有限。在本文中，我们描述并比较了从HT115细菌系统中经济（43.2欧元/毫克）和大规模（毫克级别）生产高质量dsRNA的方法。我们用靶向特异性基因的构建体以及作为非内源对照的基因（）转化细菌。首先，我们比较了四种细胞裂解处理（加热、溶菌酶消化、超声处理和对照方案）的总RNA提取产量。其次，我们评估了这些提取的dsRNA的质量和纯度。第三，我们比较了从粗RNA提取物中进一步纯化dsRNA的方法。最后，我们证明了所产生的dsRNA在鳞翅目细胞系中诱导基因敲低的效率。本文的见解和结果将使研究人员能够进行原本成本过高的基因敲低研究，并大大缩短常规或大规模使用的dsRNA底物的生产时间。
