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首页> 外文期刊>Journal of Huazhong University of Science and Technology >Knocking-down of Nogo-A Gene Expression in PC12 Cell Line by Plasmid-based RNAi
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Knocking-down of Nogo-A Gene Expression in PC12 Cell Line by Plasmid-based RNAi

机译：基于质粒的RNAi敲低PC12细胞株中Nogo-A基因表达

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摘要

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.
机译：为了研究Nogo-A shRNA对PC12细胞系的抑制作用，设计并合成了Nogo-A shRNA（短发夹RNA或shRNA）。通过基因克隆技术将退火后的shRNA模板插入含有增强的绿色荧光蛋白（EGFP）基因的质粒pGenesil-1中，以产生真核表达载体。 lipofecamine2000将重组质粒转染到PC12细胞中，转染48 h后，通过RT-PCR和Western blotting检测Nogo-A基因的mRNA和蛋白表达水平。基因测序表明成功构建了Nogo-A shRNA真核表达载体。空载体转染组的Nogo-A mRNA和蛋白表达水平与对照组相比无明显变化（P> 0.05），shRNA转染组的表达水平显着下降（P <0.05）。结论：pGenesil-1 / Nogo-AshRNA重组质粒可有效抑制PC12细胞中Nogo-A基因的表达。

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