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Published on: March 29, 2019
[Construction and screening of neurite outgrowth inhibitory 66 eukaryotic expression vectors]
Ruofei Li1, Xiaoqian Dang, Lianwei Li
1Department of Orthopedics, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P.R. China.
Objective:
To construct and screen neurite outgrowth inhibitory 66-small interfering RNA (nogo66-siRNA) eukaryotic expression vectors of effective interference, so as to lay a foundation for further reconstruction of related viral vector.
Methods:
The nogo66-siRNA fragments were designed and cloned into pGenesil-1.1, 4 plasmids of pGenesil-nogo66-siRNA-1, pGenesil-nogo66-siRNA-2, pGenesil-nogo66-siRNA-hk, and pGenesil-nogo66-siRNA-kb were obtained, sequenced and identified, then were transfected into C6 cell line. The transfection efficiency was measured by fluorescence microscope. RT-PCR and Western blot were used to detect the expression of nogo gene and select the plasmid of effective interference.
Results:
DNA sequencing results showed interference sequences were correct. The bands of 800 bp and 4.3 kb were detected when pGenesil-nogo66-siRNAs were digested by Kpn I/Xho I. The expression of green fluorescent protein could be detected under fluorescence microscope, and the transfection efficiency was about 73%. RT-PCR and Western blot results showed that compared to non-transfected cells, the transfection of pGenesil-nogo66-siRNA-1 made the expression of nogo gene decline 22% and the expression of nogo protein decline 73%; the transfection of pGenesil-nogo66-siRNA-2 made the expression of nogo gene decline 28% and the expression of nogo protein decline 78%; the differences were significant (P < 0.05); and the transfection of pGenesil-nogo66-siRNA-hk and pGenesil-nogo66-siRNA-kb did not make the expressions of nogo gene and nogo protein decrease significantly (P > 0.05).
Conclusion:
Nogo66-siRNA eukaryotic expression vector is successfully constructed, it lays an experimental foundation for repair of spinal cord injury.
