Abstract:

BACKGROUND: Recent studies suggest that the chondroitin sulfate peoteoglycan (NG2) is involved in physiological and pathological function in the central nervous system. Lentiviral vector can infect cells in both dividing phase and non-dividing phase, and can be stable expressed in cells with high efficiency.
OBJECTIVE: To construct a lentiviral vector for RNA interference (RNAi) of rat NG2 gene and to detect its effect of gene silence in C6 cells.
METHODS: Towards rat NG2 gene sequences, a pair of complementary small hairpin RNA (shRNA) oligonucleotides were designed, synthesized, annealed and inserted into pFU-GW-RNAi vector digested by Hpa Ⅰ and Xho Ⅰ. The recombinant plasmid was identified by PCR and DNA sequencing. The recombinant plasmid, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells to obtain lentivirus particles. Viral titer was then determined. The lentivirus particles were transmitted into C6 cells. Then, western blot was performed to determine the expression level of the NG2 protein.
RESULTS AND CONCLUSION: The PCR identification and DNA sequencing showed that the fragment and nucleotides were in accordance with the target sequence and the shRNA sequence was successfully inserted into the pFU-GW-RNAi vector. The titer of concentrated virus was 8×1011 TU/L. Compared with the C6 cells infected with LV-Con-RNAi or uninfected, the cells infected with LV-NG2-RNAi at MOI of 50 showed significant suppression in the expression of NG2, it could achieve 100% interference efficiency (P < 0. 05). The results demonstrate a lentiviral shRNA expression vector targeting the NG2 gene is successfully constructed and it can knockdown the expression of NG2 in C6 cells.