Xiong Nanxiang, Pu Jianzhang, Zhao Hongyang, Su Qun, Jiang Xiaobing, Yao Dongxiao
Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2007 Aug;27(4):433-6. doi: 10.1007/s11596-007-0421-6.
To study the inhibitory effect of Nogo-A shRNA on cell line PC12, the Nogo-A shRNA (short hairpin RNA, or shRNA) was designed and synthesized. The annealed shRNA template was inserted into plasmid pGenesil-1 containing enhanced green fluorescent protein (EGFP) gene by gene cloning technique to generate eukaryotic expression vector. The recombinant plasmid was transfected into PC12 cells by lipofecamine2000 and the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blotting 48 h after the transfection. Gene sequencing showed that that the Nogo-A shRNA eukaryotic expression vector was successfully constructed. No significant change was found in the Nogo-A mRNA and protein expression level in empty vector-transfected group as compared with controls (P>0.05), while the expression level in shRNA-transfected group decreased significantly (P<0.05). It is concluded that the pGenesil-1/Nogo-AshRNA recombinant plasmid can effectively suppress the expression of Nogo-A gene in PC12 cells.
为研究Nogo - A短发夹RNA（shRNA）对PC12细胞系的抑制作用，设计并合成了Nogo - A shRNA（短发夹RNA）。通过基因克隆技术将退火后的shRNA模板插入含有增强型绿色荧光蛋白（EGFP）基因的质粒pGenesil - 1中，构建真核表达载体。采用脂质体2000将重组质粒转染至PC12细胞，转染48小时后，通过逆转录聚合酶链反应（RT - PCR）和蛋白质免疫印迹法（Western blotting）检测Nogo - A基因的mRNA和蛋白质表达水平。基因测序结果表明成功构建了Nogo - A shRNA真核表达载体。与对照组相比，空载体转染组的Nogo - A mRNA和蛋白质表达水平无显著变化（P>0.05），而shRNA转染组的表达水平显著降低（P<0.05）。结论：pGenesil - 1/Nogo - AshRNA重组质粒能有效抑制PC12细胞中Nogo - A基因的表达。
