Construction Of Lentivirus Vcctor Containing Human Osteoprotegerin And Its Expression In Rat Hone Mesenchymal Stem Cells

ObjectiveTo construct a recombiant lentivirus vector of osteoprotegerin (OPG) and detect the expression of OPG in infected rat bone mesenchymal stem cells.MethodsOPG gene from the cDNA library were extracted by Polymerase Chain Reaction (PCR),The OPG genes were connected into lentiviral vectors pGC-FU-RFP which was linearized by Age I enzyme to produce recombiant lentivirus vector called as pGC-FU-OPG-RFP, then packaged by293T cells,the virus supernant congtaining LV-OPG-RFP was harvested,concentrated and titrated.The rat BMSCs were transfected with recombiant lentivirus LV-OPG-RFP at the most appropriate MOI,the mRNA and protein expression of OPG were detected by RT-PCR and Western blot.Results1n LV-OPG-RFP was recombined successfully and the titer reached2x108TU/ml.2-, The rat bone mesenehymal stem cells were transfected by LV-OPG-RFP with MOI:30,50,100. Red fluorescent protein were detected by Inverted fluorescent microscope in all transfected cells. The transfection efficiency respectively is20.2%,49.7%,89.0%.3-, The efficiency of infection was89.0%,which was get after LV-OPG-RFP infected rat BMSCs at the most appropriate MOI=100.The expression of OPG gene was confirmed by RT-PCR and Western blot. ConclusionLentivirus vector containing human OPG gene was constructed successfully,can transfected efficiently into rat BMSCs,and the infected rat BMSCs can effectively express OPG.