Li Ruofei, Dang Xiaoqian, Li Lianwei, Wang Kunzheng, Bai Chuanyi, Bao Lizhong, Lou Gaojie
Department of Orthopedics, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P.R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Feb;24(2):185-9.
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.
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.
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).
Nogo66-siRNA eukaryotic expression vector is successfully constructed, it lays an experimental foundation for repair of spinal cord injury.
构建并筛选有效干扰神经突生长抑制因子66（nogo66）的小干扰RNA（nogo66-siRNA）真核表达载体，为进一步构建相关病毒载体奠定基础。
设计nogo66-siRNA片段并克隆至pGenesil-1.1，获得pGenesil-nogo66-siRNA-1、pGenesil-nogo66-siRNA-2、pGenesil-nogo66-siRNA-hk和pGenesil-nogo66-siRNA-kb 4种质粒，进行测序和鉴定，然后转染至C6细胞系。通过荧光显微镜检测转染效率。采用RT-PCR和Western blot检测nogo基因的表达，筛选有效干扰质粒。
DNA测序结果显示干扰序列正确。pGenesil-nogo66-siRNAs经Kpn I/Xho I酶切后，检测到800 bp和4.3 kb条带。荧光显微镜下可检测到绿色荧光蛋白表达，转染效率约为73%。RT-PCR和Western blot结果显示，与未转染细胞相比，转染 pGenesil-nogo66-siRNA-1使nogo基因表达下降22%，nogo蛋白表达下降73%；转染pGenesil-nogo66-siRNA-2使nogo基因表达下降28%，nogo蛋白表达下降78%；差异有统计学意义（P < 0.05）；转染pGenesil-nogo66-siRNA-hk和pGenesil-nogo66-siRNA-kb未使nogo基因和nogo蛋白表达显著下降（P > 0.05）。
成功构建了nogo66-siRNA真核表达载体，为脊髓损伤修复奠定了实验基础。
