Kamath Ravi S, Ahringer Julie
Wellcome Trust/Cancer Research UK Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.
Methods. 2003 Aug;30(4):313-21. doi: 10.1016/s1046-2023(03)00050-1.
In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.
在秀丽隐杆线虫中，引入双链RNA（dsRNA）会导致具有相应序列的内源基因特异性失活；该技术被称为RNA干扰（RNAi）。此前已有研究表明，RNAi可通过将dsRNA直接显微注射到成年雌雄同体线虫中、将线虫浸泡在dsRNA溶液中或让线虫食用表达靶基因dsRNA的大肠杆菌来实现。我们开发了一种简单优化的方案，利用dsRNA引入的第三种方式——通过喂食进行RNAi，该方法能够对线虫的基因功能进行快速有效的分析。此外，我们构建了一个细菌菌株文库，该文库对应于秀丽隐杆线虫中估计的19000个预测基因中的约86%，并利用它对线虫的基因功能进行全基因组分析。这个文库是公开可用的、可重复使用的资源，可用于快速大规模的RNAi实验。我们已使用这个文库对线虫的基因功能进行全基因组分析。在此，我们描述用于构建细菌文库以及使用通过喂食进行RNAi在线虫中进行高通量筛选的方案。
