Heritable gene silencing in Drosophila using double-stranded RNA
Heritable gene silencing in Drosophila using double-stranded RNA
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DOI：
10.1038/78531
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发表时间：
2000-08-01
影响因子：
46.9
通讯作者：
Carthew, RW
中科院分区：
文献类型：
--
作者：
Kennerdell, JR;Carthew, RW
rna介导干扰（RNAi）是最近发现的一种测定许多生物基因功能的方法，包括植物1、线虫2、果蝇3、4、斑马鱼5和小鼠6。将单个基因对应的双链RNA （dsRNA）注射到生物体中，可使特定基因2,3,4,5,6的表达沉默。受影响细胞中mRNA的快速降解会阻断基因表达2。尽管RNAi有望成为功能基因组学的工具，但注射dsRNA会短暂干扰基因表达，并且不稳定遗传2,3,5,6。因此，使用RNAi来研究发育后期的基因功能一直受到限制。对产后个体作出反应的疾病模型的发展尤其成问题。为了在果蝇中规避这一问题，我们开发了一种将dsRNA表达为扩展发夹环RNA的方法。这种方法最近已经成功地在秀丽隐杆线虫中产生了RNAi。发夹RNA是由转基因表达的，在受控的时间和空间模式下表现出双体对称性。我们报道，稳定遗传的转基因赋予胚胎基因表达的特定干扰，并产生成人结构，如翅膀，腿，眼睛和大脑的组织。因此，RNAi可以应用于果蝇晚作用基因功能的研究。这种方法在果蝇和秀丽隐杆线虫上的成功表明，类似的方法可能被证明对研究转基因技术可用的高等生物的基因功能有用。
RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants 1, nematodes 2, Drosophila 3, 4, zebrafish 5, and mice 6. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene 2, 3, 4, 5, 6. Rapid degradation of mRNA in affected cells blocks gene expression 2. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited 2, 3, 5, 6. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans 7. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.