University of Cincinnati, Blue Ash College, Biology Dept. 9555 Plainfield Rd., Blue Ash, Ohio; Department of Biology, Duke University, Durham, NC, USA.
Department of Biology, Duke University, Durham, NC, USA.
Dev Biol. 2024 Dec;516:59-70. doi: 10.1016/j.ydbio.2024.08.002. Epub 2024 Aug 3.
Dicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchin Lytechinus variegatus (Lv). Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods are described for the design of specific DsiRNAs that lead to destruction of targeted mRNA. DsiRNAs directed against pks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNA in situ analysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition of nodal and lefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.
双链干扰 RNA（DsiRNAs）通过 RNA 诱导沉默复合物（RISC）来破坏靶向转录本，这个过程被称为 RNA 干扰（RNAi）。该过程在真核生物中普遍存在。在这里，我们报告了 DsiRNA 在海胆 Lytechinus variegatus（Lv）胚胎中的应用。特定的敲低与已知的 morpholino 和抑制剂敲低表型相同，DsiRNA 是 morpholino 的一种有用替代品。本文描述了设计特定 DsiRNA 的方法，这些 DsiRNA 可导致靶向 mRNA 的破坏。针对 pks1 的 DsiRNA 是一种酶，它是色素产生所必需的，这表明 RNA 原位分析和 qPCR 可成功监测 DsiRNA 干扰，以确定靶向 mRNA 的相对破坏情况。基于 DsiRNA 的敲低与基于 morpholino 和药物的 nodal 和 lefty 抑制作用相似。其他敲低表明 RISC 在发育早期以及在原肠胚形成完成数小时后首次转录的基因中起作用。因此，DsiRNA 可有效地介导海胆胚胎中靶向 mRNA 的破坏。与海胆中广泛使用的其他方法相比，该方法在成本和易于采购方面具有显著优势，并且在操作、注射和敲低验证方面具有相当大的实验优势。
