Knocking-down of Nogo-A gene expression in PC12 cell line by plasmid-based RNAi.
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.
Xiong
Nanxiang
N
Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. mozhuoxiong@163.com
Pu
Jianzhang
J
Zhao
Hongyang
H
Su
Qun
Q
Jiang
Xiaobing
X
Yao
Dongxiao
D
D000818:Animals; D003001:Cloning, Molecular; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D009185:Myelin Proteins / Q000235:genetics* / Q000378:metabolism; D000070798:Nogo Proteins; D016716:PC12 Cells; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D051381:Rats; D014162:Transfection