Nogo受体shRNA慢病毒载体在大鼠神经干细胞中的表达

Objective: To realize a stable knockdown of NgR in primary cultured rat neural stem cells by lentivirus mediated shRNA. Methods: Lentiviral vector expressing shRNA targeting at NgR was designed and sythesized. After transfection in 293T cells, the virus granules was packeged and collected. Rat spinal neural stem cell was isolated and cultured, followed by lentivirus transfection at different MOIs. The best MOI was determined by flow cytometry detecting the expression of GFP. After hygromycin screening and monoclonal culture, the knockdown effeciency on NgR was determined by Western blot. The stability of the knockdown effect on NgR was detected by quantitative real-time PCR by using NSC on different culture days after transfection. Result: Neural spheres were observed under light microscope 6 days after isolation. The best MOI was 10 as detected by flow cytometry. The expression of NgR was knocked down by 80% by the lentiviral shRNA, which is stable and can be maitained at 70-80% in 7-28 d after infection. Conclusion: The shRNA mediated by lentivirus can stably interfere the expression of NgR in primary cultured rat neural stem cells and is hopefully to be used in future study on NSC transplantation after spinal cord injury.