Inducing gene expression by targeting promoter sequences using small activating RNAs.
Inducing gene expression by targeting promoter sequences using small activating RNAs.
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DOI：
10.14440/jbm.2015.39
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发表时间：
2015-03-11
期刊：
影响因子：
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通讯作者：
Li, Long-Cheng
中科院分区：
文献类型：
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作者：
Wang, Ji;Place, Robert F;Li, Long-Cheng
由外源核酸序列组成的载体系统仍然是特定基因异位表达的标准。这类系统提供了强大的过表达，但也有固有的缺陷，如繁琐的构建过程，排除了对基因功能重要的序列(如内含子和非翻译区)，以及与使用病毒载体相关的宿主基因组的潜在插入突变。我们和其他人最近报道了短双链RNA(DsRNAs)可以通过靶向启动子序列来诱导内源基因的表达，这种现象被称为RNA激活(RNAa)，这种dsRNAs被称为小激活RNAs(SarNas)。到目前为止，RNAa已被成功地用于诱导不同基因的表达，如肿瘤抑制基因。在这里，我们描述了一种详细的靶标选择和dsRNA设计方案，以及相关的实验，以促进培养细胞中的RNAa。这一技术可用于选择性激活内源性基因表达，用于研究基因功能、询问分子途径和重新编程细胞命运。
Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such as the tedious process of construction, excluding sequences (e.g. introns and untranslated regions) important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs) can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa) and such dsRNAs are termed small activating RNAs (saRNAs). To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.