NS特异性siRNA真核表达载体的构建

THE GENERATION OF EUKARYOTIC EXPRESSION VECTOR OF siRNA SPECIFIC FOR NUCLEOSTEMIN

目的 :构建nucleostemin(核干细胞因子 ,NS)特异性siRNA真核表达载体。方法 :以小鼠组织基因组DNA为模板 ,PCR方法克隆小鼠编码U6snRNA基因的启动子 ,将NS -siRNA表达序列、PolⅢ识别的终止信号与U6启动子连接起来 ,并将其插入 pcDNA4 /C载体中。 结果 :(1 )从小鼠基因组DNA中克隆到编码U6snRNA基因的启动子 ,(2 )构建了含有U6启动子、NS -siRNA表达序列和PolⅢ识别终止信号的NS -siRNA表达的片段 ,(3)将NS -siRNA表达片段插入了 pcDNA4 /C真核表达载体。 结论 :构建了针对NS特异性的siRNA真核表达载体 pcDNA4 /C -NS -Silencer。

Objective: To generate eukaryotic expression vector of siRNA specific for nucleostemin. Methods: Genomic DNA of mouse-tails was isolated, and it was subsequently used as the template of PCR to clone the genomic gene promoter of U6 snRNA. U6 promoter was linked with the sequence coding NS-siRNA and the termination signal which RNA polymerase Ⅲ recognized. Finally the whole frame coding NS-siRNA was inserted into pcDNA4/C vector. Results:(1)Succeeded in cloning the promoter of mouse U6 snRNA.(2)Generated the whole NS-siRNA expression frame with promoter of U6 snRNA and the termination signal of RNA polymeraseⅢ recognized. (3) The whole frame coding NS-siRNA was inserted into the eukaryotic expression vector pcDNA4/C.Conclusion:Eukaryotic expression vector of siRNA specific for nucleostemin was generated.