Design and cloning of lentiviral vectors expressing small interfering RNAs
Design and cloning of lentiviral vectors expressing small interfering RNAs
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DOI：
10.1038/nprot.2006.36
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Verma, Inder M.
中科院分区：
文献类型：
--
作者：
Tiscornia, Gustavo;Singer, Oded;Verma, Inder M.
RNA 干扰 (RNAi) 已成为下调基因表达的强大技术。使用polIII启动子表达小发夹RNA（shRNA），结合慢病毒载体介导的向多种细胞类型的基因递送的多功能性和稳健性，提供了在体外和体内长期下调特定靶基因的可能性。使用沉默慢载体可以快速、方便地建立稳定表达 shRNA 的细胞系（或转基因小鼠），用于分析基因产物敲低产生的表型。在这里，我们提出了两种可能的方案，描述了沉默慢病毒载体的设计和克隆。这些协议可以在三周内完成。
RNA interference ( RNAi) has emerged as a powerful technique to downregulate gene expression. The use of polIII promoters to express small hairpin RNAs ( shRNAs), combined with the versatility and robustness of lentiviral vector - mediated gene delivery to a wide range of cell types offers the possibility of long- term downregulation of specific target genes both in vitro and in vivo. The use of silencing lentivectors allows for a rapid and convenient way of establishing cell lines ( or transgenic mice) that stably express shRNAs for analysis of phenotypes produced by knockdown of a gene product. Here we present two possible protocols describing the design and cloning of silencing lentiviral vectors. These protocols can be completed in less than 3 weeks.