Construction Of Lenti-HIF-1α And Detection Of The Target Gene Expression In BMSCs

Objective To construct Lenti-HIF-lα,and detect HIF-1α expression and location in bone marrow mesenchymal stem cells （BMSCs） transduced by Lenti-HIF-la.Methods According to the wild-type human HIF-1α gene sequence （NM₀01530）, the primers were designed and PCR was amplified. The eukaryotic expression vector （pEGFP-N1-HIF-1α） was constructed by connecting the PCR products of the target gene to the vector pEGFP-N1, and selecting the positive clones for sequencing. To identify the recombinant plasmid of HIF-la, the target gene PCR product and the purpose of vector pEGFP-N1were digested with Nhe1and BamHI, and the recombinant vector pEGFP-N1-HIF-1α colonies were identified by PCR with gel electrophoresis. Recombiting the target sequence into the lent viral vector plenti6.3V5-DEST by using the LR recombination system, and Lenti-HIF-la （control group, Lenti-LacZ） was constructed.293T cells were transfected by using pEGFP-N1-HIF-1α and packaging quality. The virus were packaged and the stock solution of virus was collected to be concentrated with ultracentrifugation. To observed the number of fluorescent cells under fluorescence microscope, and detect the virus titer. After lentiviral titer is detected, BMSCs were transduced by Lenti-HIF-la. The RNA was extracted in the0d.1d,4d,7d.14d and21d after being transfected, and then the expression of target genes was detected and analyse by qPCR. After BMSCs had been transfected successfully, the BMSCs were immunofulorescenced.4%paraformaldehyde, washed with PBS. be closed30min in blocking solution, then add to the primary antibody and secondary antibody, join the nuclear dye. After washing with PBS, observing the location of HIF-1α in BMSCs under confocal microscope.Results The sequence of plasmid cloning was the same with the sequence in GenBank. It confirmed that the eukaryotic expression vector pEGFP-N1-HIF-1α was successfully constructed. The target gene has been cloned into recombinant plasmid by proving with digesting the colony of the recombinant vector. After BMSCs transduced Lenti-HIF-1a0d,1d,4d,7d,14d and21d, the results of qPCR showed that the over-expression of HIF-la was detected in the4d, and continued until the21d under inverted fluorescence microscope, the14d arrived the peak level expression of mRNA. Immunohistochemical results showed that the target gene is located in the nucleus of BMSCs.Conclusion We successfully constructed the Lenti-HIF-la, BMSCs were transfected with Lenti-HIF-1α after being cultured, and target gene located in the nucleus of BMSCs. This study will lay the foundation for experimental studies of bone defect repair using HIF-1α-mediated BMSCs in the future.