Reverse genetics in the tidepool: knockdown of target gene expression via RNA interference in the copepod Tigriopus californicus

Reverse genetic tools are essential for characterizing phenotypes of novel genes and testing functional hypotheses generated from next-generation sequencing studies. RNA interference (RNAi) has been a widely used technique for describing or quantifying physiological, developmental or behavioural roles of target genes by suppressing their expression. The marine intertidal copepod Tigriopus californicus has become an emerging model for evolutionary and physiological studies, but this species is not amenable to most genetic manipulation approaches. As crustaceans are susceptible to RNAi-mediated gene knock-down, we developed a simple method for delivery of gene-specific double-stranded RNA that results in significant suppression of target gene transcription levels. The protocol was examined on five genes of interest, and for each, at least 50% knock-down in expression was achieved. While knock-down levels did not reach 100% in any trial, a well-controlled experiment with one heat-shock gen...

反向遗传学工具（reverse genetic tools）对于新基因的表型表征，以及验证由下一代测序（next-generation sequencing）研究所产生的功能假说而言，是不可或缺的研究手段。RNA干扰（RNA interference, RNAi）是一项应用广泛的技术，通过抑制靶基因的表达，来阐明或量化其在生理、发育及行为层面的功能角色。海洋潮间带桡足类物种加州虎斑猛水蚤（Tigriopus californicus）已逐渐成为进化与生理学研究领域的新兴模式生物，但该物种难以适配绝大多数常规遗传操作手段。由于甲壳类动物对RNA干扰介导的基因敲低（RNAi-mediated gene knock-down）具有敏感性，我们开发了一种简便的靶基因特异性双链RNA（double-stranded RNA, dsRNA）递送方法，该方法可显著抑制靶基因的转录水平。我们针对5个目标基因对该实验方案进行了验证，每个靶基因的表达量均实现了至少50%的敲低。尽管所有实验中敲低效率均未达到100%，但针对一个热休克基因开展的严格对照实验……