Construction of RNA Interfering Lentivirus Vector Targeting Uncoupling Protein 2

Objective:To construct RNA interfering (RNAi) lentivirus vector targeting uncoupling protein 2 (UCP-2).Methods:Three specific target sequences from UCP-2 mRNA sequence (NM_011671) were designed according to the principle of siRNA design.The double-strand DNA containing interfering sequence was designed,digested and linked with pGCL-GFP.The product was transfected into competent cells and the clones were subjected to PCR identification and sequencing.The successfully constructed RNAi plasmid and UCP-2 expression plasmid were co-transfecteed into 293T cells.The transfection efficiency was observed under the fluorescence microscopy.Western blot was used to detect the expression of UCP-2 protein.RNAi plasmids with optimal interfering efficacy were screened,and co-transfected with pHelper 1.0 and Helper 2.0 into 293T cells to form lentivirus.Results:PCR and sequencing revealed that RNAi plasmids to three targets were successfully constructed.The optimal interfering targets were selected by Western blot.The lentivirus with a titer of 5×108 TU/ml was successfully packed.Conclusion:RNAi lentivirus vector of UCP-2 was constructed successfully,which provides a foundation for targeting suppression of UCP-2 gene expression to increase the success rate of fatty liver transplantation.