RNA interference by short hairpin RNAs expressed in vertebrate cells.
RNA interference by short hairpin RNAs expressed in vertebrate cells.
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DOI：
10.1385/1-59259-750-5:255
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发表时间：
2004-01-01
期刊：
影响因子：
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通讯作者：
Conklin, Douglas S
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文献类型：
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作者：
Hannon, Gregory J;Conklin, Douglas S
RNA干扰（RNAi）是目前公认的一种抑制基因表达的通用方法。当双链RNA（dsRNA）被引入细胞时，会干扰同源基因的表达，破坏它们的正常功能。在哺乳动物中，合成的短干扰RNA（siRNA）的瞬时递送（其类似于标准双链RNAi触发物的加工形式）在沉默哺乳动物基因中是有效的。然而，与沉默效应的转移效率和持续时间相关的问题限制了siRNA在哺乳动物中的应用范围。siRNA的这些缺点已经通过来自DNA载体的短发夹RNA（shRNA）的细胞表达来解决。shRNA在功效和机制方面与siRNA无法区分，但可以在细胞内从标准哺乳动物表达载体产生。通过这种方式，shRNA表达使得可以产生连续的细胞系和转基因动物，其中靶基因的抑制通过RNAi稳定地维持。因此，可以在哺乳动物中进行的基于RNAi的基因功能分析的类型已经大大扩展。我们描述了用于构建和转移稳定的shRNA表达载体的方法，所述稳定的shRNA表达载体适合于在体外或体内在哺乳动物细胞中产生功能缺失等位基因。
RNA interference (RNAi) is now established as a general method to silence gene expression in a variety of organisms. Double-stranded RNA (dsRNA), when introduced to cells, interferes with the expression of homologous genes, disrupting their normal function. In mammals, transient delivery of synthetic short interfering RNAs (siRNAs), which resemble the processed form of standard double stranded RNAi triggers, is effective in silencing mammalian genes. Issues related to transfer efficiency and duration of the silencing effect, however, restrict the spectrum of the applications of siRNAs in mammals. These shortcomings of siRNAs have been solved by the cellular expression of short hairpin RNAs (shRNAs) from DNA vectors. shRNAs are indistinguishable from siRNAs in terms of efficacy and mechanism but can be produced within cells from standard mammalian expression vectors. In this way, shRNA expression makes possible the creation of continuous cell lines and transgenic animals in which suppression of a target gene is stably maintained by RNAi. As a result, the types of RNAi-based gene function analysis that can be carried out in mammals have been greatly expanded. We describe methods for the construction and transfer of stable shRNA expressing vectors suitable for generating loss of function alleles in mammalian cells in vitro or in vivo.