| 利用双萤光素酶报告基因系统筛选有效的siRNA |
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| 引用本文： | 单志新,林秋雄,谭虹虹,邓春玉,刘晓颖,肖定璋,余细勇. 利用双萤光素酶报告基因系统筛选有效的siRNA[J]. 南方医科大学学报, 2009, 29(8): 1577 |
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| 作者姓名： | 单志新 林秋雄 谭虹虹 邓春玉 刘晓颖 肖定璋 余细勇 |
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| 作者单位： | 广东省人民医院医学研究中心/广东省心血管病研究所,广东,广州,510080;广东省人民医院医学研究中心/广东省心血管病研究所,广东,广州,510080;广东省人民医院医学研究中心/广东省心血管病研究所,广东,广州,510080;广东省人民医院医学研究中心/广东省心血管病研究所,广东,广州,510080;广东省人民医院医学研究中心/广东省心血管病研究所,广东,广州,510080;广东省人民医院医学研究中心/广东省心血管病研究所,广东,广州,510080;广东省人民医院医学研究中心/广东省心血管病研究所,广东,广州,510080 |
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| 基金项目： | 国家自然科学基金，广东省自然科学基金 |
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| 摘 要： | 目的 建立一种利用双萤光素酶报告基因系统筛选能有效抑制目的 基因表达的小干扰RNA(siRNA)的方法.方法 以绿色荧光蛋白(GFP)基因为研究对象,构建3个候选的靶向GFP的siRNA表达质粒pSi-GFPsiRNA1、pSi-GFPsiRNA2、pSi-GFPsiRNA3和阴性对照pSi-Negative.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的GFP与萤光素酶基因(LUC)的融合表达载体pGL3-GFPf.将包含3个GFPsiRNA靶序列的GFP片段插入到改建过的pGL3-promoter载体上LUC的3非翻译区(UTR),构建pGL3-GFPp.将GFP siRNA表达质粒联同内参照质粒pRL-TK分别与pGL3-GFPf、pGL3-GFPp共转化HEK293细胞.利用双萤光素酶检测试剂盒测定各组细胞中萤光素酶的活力水平,用荧光定量PCR检测各组细胞中GFP mRNA的表达水平.结果 同对照组相比,与pGL3-GFPf共转化GFP siRNA 表达质粒的各组细胞中萤火虫萤光素酶/海肾萤光素酶比值明显降低,其中GFPsiRNAl表达组中降低最显著(P<0.01);定量PCR检测也显示,GFPsiRNA1表达组中GFP mRNA的表达水平降低最明显(19<0.01).双萤光素酶活性检测和定量PCR结果还显示,与pGL3-GFPp共转化GFP siRNA表达质粒的各组细胞中,GFPsiRNA1抑制GFP的表达最显著(P<0.01).结论 本文建立了通过双萤光素酶活性检测来筛选有效的siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案.Abstract：Objective To establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system. Methods Based on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR. Results In the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously,and the reduction was more significant in cells transfected with GFPsiRNAl compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNAl as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).Conclusion The dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.
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| 关 键 词： | 双萤光素酶报告基因系统 RNA干扰 荧光定量PCR 绿色荧光蛋白 |
An efficient method for screening effective siRNAs using dual-luciferase reporter assay system |
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| SHAN Zhi-xin,LIN Qiu-xiong,TAN Hong-hong,Deng Chun-yu,LIU Xiao-ying,XIAO Ding-zhang,YU Xi-yong. An efficient method for screening effective siRNAs using dual-luciferase reporter assay system[J]. Journal of Southern Medical University, 2009, 29(8): 1577 |
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| Authors: | SHAN Zhi-xin LIN Qiu-xiong TAN Hong-hong Deng Chun-yu LIU Xiao-ying XIAO Ding-zhang YU Xi-yong |
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| Affiliation: | SHAN Zhi-xin,LIN Qiu-xiong,TAN Hong-hong,Deng Chun-yu,LIU Xiao-ying,XIAO Ding-zhang,YU Xi-yong Research Center of Medical Sciences,Guangdong Provincial People's Hospital/Guangdong Provincial Cardiovascular Diseases Institute,Guangzhou 510080,China |
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| Abstract: | Objective To establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system. Methods Based on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with t... |
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| Keywords: | dual-luciferase reporter assay system RNA interference real-time quantitative PCR green fluorescence protein |
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