Zhang Gong-yuan, Zhao Guo-qiang, Yin Lei, Zhang Qin-xian
Department of Histology and Embryology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450052, China. E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2008 Mar;28(3):392-5.
To construct a eukaryotic expression vector for the small interfering RNA (siRNA) targeting nucleostemin (NS) gene.
The siRNA targeting NS gene was designed according to the sequence of NS mRNA available in GenBank. Three siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into plasmid pRNAT-U6.1 for constructing the recombinant plasmids, which were transformed into E.coli DH5alpha strain. The plasmids, after identification by PCR and DNA sequencing, were transfected into EC9706 cell line via liposome, and the mRNA and protein expressions of NS gene in the cells were determined by RT-PCR and Western blotting, respectively.
Three recombinant plasmids were identified by PCR and sequence analysis, the results of which showed correct insertion of the designed sequences in the plasmids. RT-PCR and Western blotting showed substantially decreased mRNA and protein expressions of NS gene in the transfected cells.
The recombinant plasmid expressing the siRNA targeting NS gene has been successfully constructed, which provides the basis for studying RNA interference of the NS gene.
构建针对核干细胞因子（NS）基因的小干扰RNA（siRNA）真核表达载体。
根据GenBank中NS mRNA的序列设计靶向NS基因的siRNA。获得三条siRNA序列，合成相应的cDNA并插入质粒pRNAT-U6.1中构建重组质粒，将其转化至大肠杆菌DH5α菌株。经PCR和DNA测序鉴定后的质粒通过脂质体转染至EC9706细胞系，分别用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测细胞中NS基因的mRNA和蛋白表达。
通过PCR和序列分析鉴定出三个重组质粒，结果表明设计的序列正确插入到质粒中。RT-PCR和蛋白质免疫印迹法显示转染细胞中NS基因的mRNA和蛋白表达显著降低。
成功构建了表达靶向NS基因siRNA的重组质粒，为研究NS基因的RNA干扰提供了基础。
