| Smad3基因RNAi慢病毒载体的构建与鉴定 |
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| 引用本文： | 王延洲,梁志清,刘晓芳,徐惠成.Smad3基因RNAi慢病毒载体的构建与鉴定[J].第三军医大学学报,2009,31(8). |
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| 作者姓名： | 王延洲 梁志清 刘晓芳 徐惠成 |
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| 作者单位： | 第三军医大学西南医院妇产科,重庆,400038 |
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| 摘 要： | 目的 构建Smad3基因RNAi慢病毒载体.方法 针对已经筛选确定的Smad3基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经Age I和EcoR I双酶切后的pGCSIL-GFP载体连接产生GC-shSmad3慢病毒载体,PCR筛选阳性克隆,测序鉴定.用GC-shSmad3、pHelper 1.0载体和pHelper 2.0载体共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度.结果 PCR和测序证实,构建出了Smad3 shRNA的慢病毒载体GC-shSmad3.包装慢病毒,浓缩病毒悬液的滴度为3×103TU/ml.结论 成功构建Smad3基因RNAi慢病毒载体.
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| 关 键 词： | RNA干扰 慢病毒 |
Construction and identification of lentiviral vector encoding shRNA against Smad3 |
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| WANG Yan-zhou,LIANG Zhi-qing,LIU Xiao-fang,XU Hui-cheng.Construction and identification of lentiviral vector encoding shRNA against Smad3[J].Acta Academiae Medicinae Militaris Tertiae,2009,31(8). |
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| Authors: | WANG Yan-zhou LIANG Zhi-qing LIU Xiao-fang XU Hui-cheng |
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| Affiliation: | WANG Yan-zhou,LIANG Zhi-qing,LIU Xiao-fang,XU Hui-cheng(Department of Obstetrics and Gynecology,Southwest Hospital,Third Military Medical University,Chongqing 400038,China) |
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| Abstract: | Objective To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting Smad3 gene.Methods The targeting sequence of Smad3 gene which can be effectively silenced in RNA inference was confirmed in our previous study.The cDNA containing both sense and antisense Oligo DNA fragments of the targeting sequence was designed,synthesized and cloned into the pGCSIL-GFP vector.The obtained lentiviral vector containing Smad3 shRNA was confirmed by PCR and sequencing.293T cells were cotransfected with l... |
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| Keywords: | Smad3 |
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