大鼠STAT3基因靶向shRNA真核表达载体的构建及鉴定  

Construction and identificaiton of rat STAT3 shRNA eukaryotic expressing vectors

作　　者：张五星[1] 周伟[1] 赵学伟[1] 丁晓然[2] 周喆[2] 张智敏[1] 周焕芝[1] 石炳毅[1] 

机构地区：[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

出　　处：《山东医药》2010年第17期13-15,共3页Shandong Medical Journal

基　　金：全军"十一五"青年基金资助项目(06Q071)

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。Objective To construct a short hairpin RNA（shRNA）-expressing plasmid vectors specific for rat signal transducers and activators of transcription 3（STAT3）.Methods According to the reported STAT3 cDNA sequence and the principles of shRNA design,4 shRNA oligoes with best inhabitation effect were designed.The 4 dsDNA templates expressing above 4 shRNA oligoes were obtained by PCR amplification of 4 pairs of specifically designed primers.The 4 dsDNA templates were inserted into pGCsi-U6 plasmid using DNA recombination recrespectively.Finally,the recombinant pGCsi-U6 plasmids were identified by PCR amplification and sequence analysis.Results The dsDNA templates expressing the 4 shRNA oligoes were inserted into the pGCsi-U6 plasmids successfully,and their sequences were identical to the designed sequences.Conclusions The designed shRNA oligoes could be inserted into the plasmid correctly,and the shRNA-expressing plasmid vectors for STAT3 are constructed successfully.

关 键 词：信号转导子及转录激活子3 短发夹RNA 质粒 RNA干扰 

分 类 号：Q344.13[生物学—遗传学]