Construction of rat CXCR4 gene lentiviral RNA interference vector and its expression in mesenchymal stem cells / 生物工程学报

ABSTRACT

To construct the lentiviral RNA interference (RNAi) vector of rat CXCR4 gene, three target sequences were selected according to rat CXCR4 mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After phosphorylation and annealing, these double-strand DNA were cloned to Bgl II and Hind III sites of pSUPER. Then the product pRiCXCR4 was confirmed by electrophoresis and sequencing. Next, CXCR4 shRNA was cloned to a transfer vector of lentivirus, pNL-EGFP, and pNL-RiCXCR4-EGFP was produced. It was confirmed by digestion and sequencing that CXCR4 shRNA expression structure was correctly cloned to pSUPER and pNL-EGFP respectively. Three plasmids, pNL-RiCXCR4-EGFP, pHELPER and pVSVG were cotransfected into 293T to package lentivirus particles. The functional titer of obtained virus was determined by flow cytometry after transduction in 293T, the resulting functional titer of unconcentrated virus and concentrated virus were 6.4 x 10(4) TU/mL and 6.9 x 10(6) TU/mL respectively. After the rat mesenchymal stem cells (rMSCs) were transduced with the constructed lentiviral vectors, real-time RT-PCR, Western blotting and flow cytometry were used to evaluate the level of CXCR4 expression. Compared with control group, the CXCR4 mRNA expression were obviously suppressed in all three experimental groups (rMSCs-CXCR4a, rMSCs-CXCR4b, rMSCs-CXCR4c), especially the expression rate in rMSCs-CXCR4b group was reduced by 95.6%. The RNAi lentivirus vector of rat CXCR4 gene has been constructed successfully. This greatly facilitates the further studies such as evaluation the role of CXCR4 in rMSCs recruitment to damaged tissue.