Hohjoh Hirohiko
National Institute of Neuroscience, NCNP, Tokyo, Japan.
Methods Mol Biol. 2010;623:67-79. doi: 10.1007/978-1-60761-588-0_4.
Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. We have developed an assay system with reporter alleles that encode the Photinus and Renilla luciferase genes carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. The assay system allows us to evaluate designed siRNAs and also short hairpin RNAs for allele-specific silencing against target mutant alleles as well as off-target silencing against corresponding wild-type alleles simultaneously in a qualitative and quantitative manner.
通过RNA干扰（RNAi）实现的等位基因特异性基因沉默在治疗上可用于特异性抑制疾病相关等位基因的表达，而不抑制相应野生型等位基因的表达。为了实现这种等位基因特异性RNAi（ASP-RNAi），赋予ASP-RNAi的小干扰RNA（siRNA）双链体的设计和评估至关重要；然而，这也很困难。我们开发了一种带有报告等位基因的检测系统，这些报告等位基因编码在其3'-非翻译区携带突变和野生型等位基因序列的萤火虫荧光素酶基因和海肾荧光素酶基因。该检测系统使我们能够以定性和定量的方式同时评估设计的siRNA以及短发夹RNA对靶突变等位基因的等位基因特异性沉默以及对相应野生型等位基因的脱靶沉默。
