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两种方法研究不同茎部长度串联shRNA表达载体对EGFP基因的干扰效应
引用本文:肖红卫,刘中华,刘西梅,郑新民,乔宪凤,李莉.两种方法研究不同茎部长度串联shRNA表达载体对EGFP基因的干扰效应[J].江西农业学报,2010,22(10).
作者姓名:肖红卫  刘中华  刘西梅  郑新民  乔宪凤  李莉
基金项目:国家自然科学基金项目,湖北省国际合作项目,动物胚胎工程及分子育种湖北省重点实验室开放课题
摘    要:目的:旨在观察不同茎部长度串联shRNA表达载体对EGFP基因的干扰作用。方法:采用体外培养的Vero细胞,选用不同茎部长度串联shRNA表达载体+EGFP表达载体+脂质体转染的方法,将构建好的干扰载体转染Vero细胞,然后使用实时荧光定量RT-PCR对其沉默效应进行定量分析。在小鼠腿部肌肉注射干扰质粒,实时荧光定量RT-PCR对其沉默效应进行定量分析。结果:转染Vero细胞时,pGenesilR21、pGenesilR27和pGenesilR29荧光表达量仅为对照质粒pEGFP-C1的4.3%、3.0%和6.9%;小鼠腿部肌肉注射干扰质粒时,pGenesilR21、pGenesilR27和pGenesilR29荧光表达量仅为对照质粒pEGFP-C1的2.5%、1.3%和5.1%。结论:在细胞和个体水平,不同茎部串联干扰载体对目的基因的抑制效应均很明显(达到90%以上),比单一位点产生的沉默效应要强许多,不同茎部长度干扰效应彼此间差异不显著。

关 键 词:shRNA表达载体  Vero细胞  脂质体  EGFP表达载体  转染

Study on Silencing Effect of Tandem shRNA Expression-vectors Embracing Different Stems on EGFP Gene by Two Methods
XIAO Hong-wei,LIU Zhong-hua,LIU Xi-mei,ZHENG Xin-min,QIAO Xian-feng,LI Li.Study on Silencing Effect of Tandem shRNA Expression-vectors Embracing Different Stems on EGFP Gene by Two Methods[J].Acta Agriculturae Jiangxi,2010,22(10).
Authors:XIAO Hong-wei  LIU Zhong-hua  LIU Xi-mei  ZHENG Xin-min  QIAO Xian-feng  LI Li
Abstract:In order to study the silencing effect of different shRNA expression-vectors on EGFP gene,in vitro cultivated Vero cells were used,different shRNA expression-vectors,EGFP expression-vectors and liposome were mixed and transfected into Vero cells,and then the fluorescence quantitative RT-PCR method was used to detect the silencing effect of shRNA expression-vectors on EGFP gene.The fluorescence quantitative RT-PCR method was also used to detect the shRNA's effect of gene silence after the plasmids were injected into the leg muscle of mouse.The results showed that:(1) The EGFP expression of pGenesilR21,pGenesilR27 and pGenesilR29 was respectively regulated down to 4.3%,3.0% and 6.9% of the control plasmid pEGFP-C1 after being transfected to Vero cells.(2) The EGFP expression of pGenesilR21,pGenesilR27 and pGenesilR29 was respectively reduced to 2.5%,1.3% and 5.1% of pEGFP-C1 after being injected into the leg muscle of mouse.These results indicated that the tandem shRNA had a significant silencing effect to EGFP gene and inhibited the gene expression down to 10%,and the different shRNA? had no significant difference in the silencing effect.
Keywords:shRNA expression vector  Vero cells  Liposome  EGFP expression vector  Transfection
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