Abstract：
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.
摘要：
Dicer底物干扰RNA（DsiRNA）使用RNA诱导的沉默复合物（RISC）通过称为RNA干扰（RNAi）的过程破坏靶向的转录本。这个过程在真核生物中普遍存在。在这里，我们报告了DsiRNA在海胆Lytechinusvariegatus（Lv）胚胎中的实用性。特异性敲除表型已知吗啉代和抑制剂敲除，和DsiRNA提供了一个有用的替代吗啉。描述了用于设计导致靶向mRNA破坏的特异性DsiRNA的方法。针对pks1的DsiRNA，pks1是一种色素产生所必需的酶，显示通过RNA原位分析和通过qPCR监测DsiRNA扰动如何成功以确定靶向mRNA的相对破坏。基于DsiRNA的敲除表型吗啡和基于药物的结节和左撇子抑制。其他敲除表明，RISC在发育早期以及在原肠胚形成完成后数小时首次转录的基因上都起作用。因此，DsiRNAs有效介导海胆胚胎中靶向mRNA的破坏。该方法在成本方面比其他广泛使用的方法具有显着的优势，易于采购，并在易于处理方面提供了相当大的实验优势，注射,和击倒验证。
