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背景与目的:RNA干扰是一种通过细胞内导入双链RNA而导致特异基因表达抑制的进化保守的转录后基因沉默现象,目前RNA干扰已成为研究基因功能的重要方法。本研究旨在利用DNA载体在细胞内产生短发夹状RNA(shorthairpinRNAs,shRNA),通过这些shRNA来诱导RNA干扰(RNAinterference,RNAi),为基因功能分析提供新的实验手段。方法:利用双荧光素酶报告系统来检测DNA载体在细胞内产生的shRNA诱导RNAi的效果。比较该载体产生的shRNA在不同条件下的RNA干扰效果。结果:DNA载体产生的shRNA在人细胞能诱导RNAi,序列特异地抑制基因表达。抑制效果与所选基因靶位点高度相关。结论:shRNA在人细胞能诱导RNA干扰,序列特异地抑制基因表达,这一方法可用于基因功能分析。
BACKGROUND & OBJECTIVE: RNAi is an evolutionarily conserved posttranscriptional gene silencing that inhibits the expression of specific genes by intracellular introduction of double-stranded RNA. At present, RNA interference has become an important method for studying gene function. The purpose of this study is to use DNA vectors to generate short hairpin RNAs (shRNAs) in cells. These shRNAs can induce RNA interference (RNAi) and provide new experimental methods for gene function analysis. Methods: Dual luciferase reporter system was used to detect the effect of shRNA induced RNAi produced by DNA vector in cells. The shRNA generated by this vector was compared for its RNA interference effect under different conditions. RESULTS: DNA vector-generated shRNAs induced RNAi in human cells and sequence-specific inhibition of gene expression. The inhibitory effect is highly correlated with the target site of the selected gene. Conclusion: shRNA can induce RNA interference in human cells and suppress gene expression sequence-specifically. This method can be used for gene function analysis.