Coupling of RNAi-mediated target downregulation with gene replacement
Coupling of RNAi-mediated target downregulation with gene replacement
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DOI：
10.1089/108729003768247619
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发表时间：
2003-01-01
期刊：
影响因子：
--
通讯作者：
Rossi, JJ
中科院分区：
文献类型：
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作者：
Kim, DH;Rossi, JJ
短干扰RNA（siRNA）介导的有害内源性转录物的敲低在遗传性疾病的治疗中具有潜在的应用。在突变体和野生型转录物不能通过siRNA彼此区分的情况下，可能需要用对siRNA活性有抗性的靶RNA的修饰形式同时进行基因置换。为了测试这种可能性，我们利用了一种有效的siRNA来敲低EGFP mRNA。在该系统中，野生型EGFP表达被siRNA抑制，而在靶区域具有密码子修饰的EGFP构建体（其在其他方面是完全功能性的）不被下调。当野生型信息的表达被抑制时，可以通过用密码子修饰的EGFP转染这些细胞来同时恢复EGFP表达。这些研究提供了一个详细的方法和系统来测试这种策略与RNA干扰（RNAi）。
Short interfering RNA (siRNA)-mediated knockdown of deleterious endogenous transcripts has potential applications for the treatment of hereditary diseases. In situations where the mutant and wildtype transcripts cannot be discriminated from one another by siRNAs, it may be necessary to simultaneously carry out gene replacement with a modified form of the target RNA that is resistant to siRNA activity. To test this possibility, we have taken advantage of a potent siRNA that knocks down EGFP mRNA. In this system, wild-type EGFP expression is suppressed by the siRNA, whereas an EGFP construct with codon modifications in the target region that is otherwise fully functional is not downregulated. When expression of the wild-type message is inhibited, EGFP expression can be simultaneously restored by transfecting these cells with the codon-modified version of EGFP. These studies provide a detailed methodology and system for testing this strategy with RNA interference (RNAi).