人Hes1-shRNA,Hes5-shRNA慢病毒表达载体的构建、包装及鉴定

目的 构建人Hes1-shRNA和Hes5-shRNA慢病毒表达载体,为Notch-Hes信号通路的相关研究奠定基础.方法 根据人Hes1,Hes5基因mRNA序列分别设计、合成多对互补的DNA单链寡核苷酸,退火后克隆至pENTR /U6入门载体.通过入门载体瞬时转染神经胶质瘤U251细胞筛选有效干扰序列.将含有效干扰序列的入门载体与pLenti6/BLOCK-iT-DEST载体进行LR重组构建Hes1-shRNA和Hes5-shRNA慢病毒表达载体,经脂质体介导入293FT细胞,包装成慢病毒.用该慢病毒感染U251细胞,Western印迹法分别检测Hes1,Hes5蛋白的表达.结果 分别构建了针对Hes1和Hes5基因的特异性shRNA慢病毒表达载体,其包装获得慢病毒可有效感染U251细胞并分别对Hes1,Hes5蛋白的表达有显著抑制作用.结论 成功构建了Hes1-shRNA和Hes5-shRNA慢病毒表达载体.

Construction,Package and Identification of Lentiviral Vectors Expressing shRNA Targeting Human Hes1,Hes5 Gene

Objective To construct lentiviral vectors expressing shRNA targeting human Hes1 and Hes5 genes to investigate Notch-Hes signal pathway.Methods Complementary single-stranded DNA oligos were designed and synthesized according to mRNA sequences of human Hes1 and Hes5.The single-stranded oligos were annealed to generate ds-oligos and then linked with linearized pENTR/U6 to obtain entry clones.The LR recombination reactions were performed between the entry clones and pLenti6/BLOCK-iT-DEST to generate lentiviral vectors expressing small hairpin RNA(shRNA)targeting Hes1 and Hes5 gene respectively.Then,the lentiviral vectors were cotransfected with viral packaging mix into 293FT cells to generate lentiviral stock.Human glioma U251 cells were infected with lentiviral stock.Expression level of Hes1 or Hes5 protein in the infected cells was determined by Western blot.Results The recombinant lentiviral vectors expressing Hes1-shRNA and Hes5-shRNA were successfully constructed.The generated lentiviral stock effectively infected U251 cells,resulting in suppression of Hes1 or Hes5 protein expression.Conclusion Lentiviral vectors expressing shRNA targeting human Hes1 and Hes5 gene were successfully constructed.