[Construction and screening of neurite outgrowth inhibitory 66 eukaryotic expression vectors].
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).


CONCLUSIONS
Nogo66-siRNA eukaryotic expression vector is successfully constructed, it lays an experimental foundation for repair of spinal cord injury.
Li
Ruofei
R
Department of Orthopedics, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P.R. China.
Dang
Xiaoqian
X
Li
Lianwei
L
Wang
Kunzheng
K
Bai
Chuanyi
C
Bao
Lizhong
L
Lou
Gaojie
G
D000818:Animals; D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors; D009185:Myelin Proteins / Q000235:genetics*; D000070798:Nogo Proteins; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D014162:Transfection
D009185:Myelin Proteins; D000070798:Nogo Proteins; D034741:RNA, Small Interfering; C000603197:Rtn4 protein, rat