Abstract:

Objective  To construct a lentiviral vector for RNA interference of rat NF-κB p65 gene and transfect it  into spinal neurons to observe its silencing efficiency.  Methods  Three siRNA sequences and a negative control sequence were designed according to NF-κB p65 gene sequence of rats. The complementary DNA containing both sense and antisense DNA oligos of the targeting sequence was synthesized and cloned into the pFU-GW vector, which were digested by BamH I and EcoR I. The recombined vector was confirmed by PCR and DNA sequencing and cotransfected with pHelper1.0 and pHelper 2.0 packaging plasmids into 293T cells by use of Lipofectamine 2000, then the virus titer was measured. Finally, the recombinant lentivirus was transfected into spinal neurons, which were divided into five groups: negative control group, LV-NC group, LV-shNF-κB p65-1group, LV-shNF-κB p65-2 group and LV-shNF-κB p65-3 group. The transfection efficiencies were observed in each group. Results  DNA sequencing Results   demonstrated that the inserted sequences were correct. The titer of virus was 108-9 TU/mL. Recombinant lentivirus could be successfully transfected into spinal neurons with the transduction rate higher than 90%. Compared with negative control or LV-NC groups, LV-shNF-κB p65-1 or LV-shNF-κB p65-2 groups showed a lower expression level of NF-κBp65 protein（all P<0. 01）, while  LV-shNF-κB p65-3 group didn’t (P>0.05). Conclusion  The lentivirus RNAi vector targeting rat spinal neuronal NF-κB p65 gene has been constructed successfully. It may down-regulate NF-κB p65 expression in spinal neurons.

Key words: Neurons, Lentiviruses, Rats, RNAi, Nuclear transcription factor-κB p65