Development of an efficient RNA interference method by feeding for the microcrustacean Daphnia.
Development of an efficient RNA interference method by feeding for the microcrustacean Daphnia.
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DOI：
10.1186/s12896-015-0209-x
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发表时间：
2015-10-07
影响因子：
3.5
通讯作者：
Patel RC
中科院分区：
文献类型：
--
作者：
Schumpert CA;Dudycha JL;Patel RC
RNA干扰（RNA interference，RNAi）是研究基因功能的重要分子工具。水蚤是一种淡水微甲壳动物，是一种新兴的模式生物，用于研究衰老、发育和生态毒理学的细胞和分子过程，特别是在环境变化的背景下。然而，尽管蚤状蚤的全基因组测序的可用性，有意义的机制的研究一直受到阻碍缺乏分子技术来改变基因表达。已经描述了通过RNAi进行基因敲低的显微注射方法，但是对高度专业化的设备以及技术专长的需要限制了该技术的更广泛应用。除了昂贵和技术上的挑战外，显微注射只能靶向在胚胎阶段表达的基因，因此很难在成年生物中实现有效的RNAi。在我们目前的研究中，我们提出了一种细菌饲养方法的RNAi在水蚤。我们使用了一种黑色的水蚤物种（Daphnia melanica），表现出深色色素沉着的目标酚氧化酶，黑色素生物合成中的关键酶。我们证明了我们的RNAi方法导致了显著的表型，并且由于RNAi，酚氧化酶mRNA表达和黑色素含量以及UV损伤后的存活率降低。总的来说，我们的研究结果建立了一种新的方法，在水蚤RNAi显着推进进一步使用水蚤作为模式生物的功能基因组学研究。我们描述的方法是相对简单的，广泛适用于成年生物体中的各种基因的敲低。
RNA interference (RNAi) is an important molecular tool for analysis of gene function in vivo. Daphnia, a freshwater microcrustacean, is an emerging model organism for studying cellular and molecular processes involved in aging, development, and ecotoxicology especially in the context of environmental variation. However, in spite of the availability of a fully sequenced genome of Daphnia pulex, meaningful mechanistic studies have been hampered by a lack of molecular techniques to alter gene expression. A microinjection method for gene knockdown by RNAi has been described but the need for highly specialized equipment as well as technical expertise limits the wider application of this technique. In addition to being expensive and technically challenging, microinjections can only target genes expressed during embryonic stages, thus making it difficult to achieve effective RNAi in adult organisms. In our present study we present a bacterial feeding method for RNAi in Daphnia. We used a melanic Daphnia species (Daphnia melanica) that exhibits dark pigmentation to target phenoloxidase, a key enzyme in the biosynthesis of melanin. We demonstrate that our RNAi method results in a striking phenotype and that the phenoloxidase mRNA expression and melanin content, as well as survival following UV insults, are diminished as a result of RNAi. Overall, our results establish a new method for RNAi in Daphnia that significantly advances further use of Daphnia as a model organism for functional genomics studies. The method we describe is relatively simple and widely applicable for knockdown of a variety of genes in adult organisms.