Cellular and Molecular Biology, The University of Southern Mississippi, Hattiesburg, MS, USA.
Methods Mol Biol. 2022;2360:253-277. doi: 10.1007/978-1-0716-1633-8_19.
RNAi is a gene-silencing mechanism conserved in the vast majority of eukaryotes. It is widely used to study gene function in animals due to the ease of eliciting gene knockdown. Beyond research applications, RNAi technology based on exogenous dsRNA is a promising candidate for next generation insect pest control. An advantage of using RNAi is that design of dsRNA essentially requires only the sequence of the target gene. The greatest challenge, however, is dsRNA delivery for large-scale insect control. Delivery methods that have widely been used are oral, injection, or via soaking. Unfortunately, each insect presents its own challenges owing to the differences in the presence of dsRNA degrading enzymes, cellular uptake efficiency, expression of core RNAi machinery, the nature of the target gene, the concentration and persistence of the dsRNA, as well as the particular way of feeding of each insect, which together cause variations in the efficiency of RNAi. In this chapter, a protocol for the synthetic production of dsRNA is described along with three methods for delivery that have been successful in one of the more problematic insects, Diaphorina citri.
RNAi 是一种在绝大多数真核生物中保守的基因沉默机制。由于易于引发基因敲低，它被广泛用于研究动物中的基因功能。除了研究应用之外，基于外源 dsRNA 的 RNAi 技术是下一代害虫防治的有前途的候选技术。使用 RNAi 的一个优势是 dsRNA 的设计实质上只需要目标基因的序列。然而，最大的挑战是 dsRNA 的大规模昆虫控制的输送。广泛使用的输送方法是口服、注射或浸泡。不幸的是，由于 dsRNA 降解酶的存在、细胞摄取效率、核心 RNAi 机制的表达、靶基因的性质、dsRNA 的浓度和持久性以及每种昆虫的特殊进食方式的差异，每种昆虫都存在自身的挑战，这共同导致了 RNAi 的效率存在差异。在本章中，描述了合成 dsRNA 的生产方案以及已在更成问题的昆虫之一柑橘木虱中成功使用的三种输送方法。
