Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.
Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.
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DOI：
10.1186/gb-2000-2-1-research0002
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发表时间：
2001
期刊：
影响因子：
12.3
通讯作者：
中科院分区：
文献类型：
--
作者：
RNAi可以通过喂养表达对应于特定基因的双链RNA的蠕虫大肠杆菌来实现。提出了一种优化的饲养方法，其导致表型至少与通过直接注射RNA产生的胚胎致死基因的表型一样强，并且对于具有胚后表型的基因更强。在秀丽隐杆线虫中，注射双链RNA（dsRNA）导致含有同源序列的基因的特异性失活，这种技术称为RNA介导的干扰（RNAi）。先前已经表明，RNAi也可以通过喂养表达对应于特定基因的dsRNA的蠕虫大肠杆菌来实现;然而，这种dsRNA引入模式通常被认为比直接注射效率低，因此使用有限，尽管它的劳动强度大大降低。在这里，我们提出了一种优化的饲养方法，其导致表型至少与直接注射dsRNA产生的胚胎致死基因一样强，并且对于具有胚胎后表型的基因更强。此外，可以滴定由进食产生的干扰效应，以揭示一系列关于给定基因功能的亚型表型信息。利用这种方法，我们在连续cosmetics上筛选了86个随机基因，并鉴定了13个新基因的功能。这些包括两个基因产生不协调的表型（以前未表征的POU同源结构域基因，ceh-6，和一个基因编码的MADS盒蛋白）和一个基因编码一种新的蛋白质，导致高发病率的男性表型。通过喂食的RNAi可以提供关于单个基因功能的重要信息，而不是通过注射提供。此外，它可以用于注射或使用突变体有时不可行的特殊应用（例如滴定，生物化学和大规模筛选）。因此，通过喂食的RNAi应该使使用基因组序列信息的新实验方法成为可能。
RNAi can be achieved by feeding worms Escherichia coli expressing dousble-stranded RNA corresponding to a specific gene. An optimized feeding method is presented that results in phenotypes at least as strong as those produced by direct injection of RNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive. Here we present an optimized feeding method that results in phenotypes at least as strong as those produced by direct injection of dsRNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. In addition, the interference effect generated by feeding can be titrated to uncover a series of hypomorphic phenotypes informative about the functions of a given gene. Using this method, we screened 86 random genes on consecutive cosmids and identified functions for 13 new genes. These included two genes producing an uncoordinated phenotype (a previously uncharacterized POU homeodomain gene, ceh-6, and a gene encoding a MADS-box protein) and one gene encoding a novel protein that results in a high-incidence-of-males phenotype. RNAi by feeding can provide significant information about the functions of an individual gene beyond that provided by injection. Moreover, it can be used for special applications for which injection or the use of mutants is sometimes impracticable (for example, titration, biochemistry and large-scale screening). Thus, RNAi by feeding should make possible new experimental approaches for the use of genomic sequence information.
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影响因子：
9.2
作者：
Hunter, CP
通讯作者：
Hunter, CP
影响因子：
9.2
作者：
Fraser, AG;James, C;Hengartner, MO
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Hengartner, MO
影响因子：
14.9
作者：
Bateman, A;Birney, E;Sonnhammer, ELL
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Sonnhammer, ELL
影响因子：
64.5
作者：
KELEHER, CA;REDD, MJ;JOHNSON, AD
通讯作者：
JOHNSON, AD
影响因子：
5.3
作者：
Ohi, R;Feoktistova, A;Gould, KL
通讯作者：
Gould, KL