大鼠STAT3基因靶向shRNA真核表达载体的构建及鉴定

作者:张五星;周伟;赵学伟;丁晓然;周喆;张智敏;周焕芝;石炳毅 刊名:山东医药 上传者:刘干斌

【摘要】目的针对大鼠信号转导子及转录激活子3(STAT3)基因序列,构建特异性短发夹状RNA(shRNA)真核表达载体。方法根据STAT3基因序列及shRNA设计原则,设计4条干扰效果最佳的shRNA,化学合成4对引物,通过PCR扩增获得dsDNA模板,采用DNA重组技术与pGCsi-U6/Hygro/GFP空载体连接,转化DH5a感受态细胞经诱导表达筛选阳性克隆子,抽提重组质粒,进行DNA测序鉴定。结果转化大肠杆菌涂布平板长出阳性菌落,测序结果与所设计的STAT3 shRNA转录模板序列一致。结论所设计的shRNA编码序列被正确地插入到质粒骨架中,成功构建了STAT3基因的shRNA表达载体。

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大鼠STAT3基因靶向shRNA真核表达载体的构建及鉴定 张五星1。周伟1。赵学伟1.丁晓然2。周酷2,张智敏1,周焕芝1,石炳毅1 (1解放军第309医院全军移植中心,北京100091;2军事医学科学院放射与辐射医学研究所) 摘要:目的针对大鼠信号转导子及转录激活子3(STAT3)基因序列,构建特异性短发夹状RNA(shRNA)真核表达载体。方法根据STA'IB基因序列及shRNA设计原则,设计4条干扰效果最佳的shRNA,化学合成4对引物,通过PCR扩增获得dsDNA模板,采用DNA重组技术与t)C,Csi-U6/Hygro/GFP空载体连接,转化DHSa感受态细胞经诱导表达筛选阳性克隆子,抽提重组质粒,进行DNA测序鉴定。结果 转化大肠杆菌涂布平板长出阳性菌落,测序结果与所设计的STAT3 shRNA转录模板序列一致。结论所设计的shRNA编码序列被正确地插入到质粒骨 架中。成功构建了STAT3基因的shRNA表达载体。 。关键词:信号转导子及转录激活子3;短发夹RNA;质粒;RNA干扰 中图分类号:Q344+.13 文献标志码:A 文章编号:1002-266X(2010)17-0013-03 Construction and identificaiton of rat STA.r3 shRNA eukaryotic expressing vectors ZHANG Wu-xingl。ZHOU Wei.ZHAO Xue—wei.DlNG Xiao-ran.ZHOU Zk,ZHANG Zhi—min。 ZHoU Huan-zhi。sm Bing-yi (1 Transplantation Center of309th Hospital ofPeople’s Liberation Army,Beijing 100091,P.R.China) Abstract:Objective To construct n 8hort hairpin RNA(shRNA)-expressing plasmid vectors specific for rat sisnal transduccm and activators of transcription 3(STAT3).Methods According to the reported STAT3 cDNA sequence and the pHncipl∞of shRNA design。4 shRNA oligoes with best inhabitation effect were designed.The 4 dsDNA templates嘶pressing above 4 shRNA oligoes were obtained by PCRamplification d4 pai礴of specifically designed primers.The 4 d8m NA templates were inserted into pGCsi—U6 phsmid using DNA recombination recrespectively.Finally,the recombinant pGCsi·U6 phsmids we弛identified by PCR amplification and sequence analysis.Results The dsDNA templates expressing the 4 shRNA oligoes were inserted into the pC

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