Timmons L, Court D L, Fire A
Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210, USA.
Gene. 2001 Jan 24;263(1-2):103-12. doi: 10.1016/s0378-1119(00)00579-5.
Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
由双链RNA介导的基因干扰（RNA干扰）已成为分析秀丽隐杆线虫基因功能的一项重要工具。在此，我们报告了一种利用细菌递送双链RNA来高效诱导RNA干扰的方法。该方法利用了缺乏核糖核酸酶III（RNaseIII）的细菌，这种酶通常会降解细菌细胞中的大部分双链RNA。对缺乏RNaseIII的细菌进行改造，使其能够大量产生特定的双链RNA片段。当将这些经过改造的细菌喂食给秀丽隐杆线虫时，发现它们能产生一群受到RNA干扰影响的动物，其表型在表达程度上与相应的功能丧失型突变体相当。我们发现该方法在诱导晚期幼虫和成年雌雄同体的非神经组织的RNA干扰方面最为有效，而在神经系统、幼虫早期阶段以及雄性个体中效果则有所降低。通过持续喂食，细菌诱导的RNA干扰表型可以在几代过程中得以维持，这便于对特定基因干扰的生物学后果以及持续暴露于双链RNA的影响进行评估。
