Optimize multiplicity of infection and selected concentration of antibiotics in construction of monoclonal stable cell line by lentivirus vector-mediated RNA interence silenced gene SGMS2 / 中国生化药物杂志

ABSTRACT

Objective To optimize multiplicity of infection ( MOI) and antibiotics ( blasticidin) concentration selecting BSD gene in construction of monoclonal stable cell line by lentivirus vector-mediated RNA interence silenced gene SGMS2.Methods The INS-1 cells were transfected by fluorescence labeled negative control SGMS2-siRNA lentivirus at MOI of 0, 10, 30, 60 and 120 TU number/cell.The cells were photographed under fluorescent microscopy after 72 h cultivation, then fluorescence ratio and apoptosis rate were calculated to determine optimal MOI.The INS-1 cells were treated by blasticidin with different concentrations of 0, 1, 2, and 3 μg/mL, and the apoptosis rate was observed to acquire optimal concentration of antibiotics.The INS-1 cells were transfected by negative control SGMS2-siRNA lentivirus and SGMS2-siRNA lentivirus (virus titer1 ×108TU/mL) at optimal MOI and positive-transfected cells were selected by blasticidin at optimal concentration, then mixed cell lines were acquired.The monoclonal cell line was constructed at fluorescence ratio of 90%.Results The optimal MOI was 60 with 100% fluorescence ratio, less than 0.5% apoptosis rate and keep original cellular morphology.The optimal concentration of blasticidin was 2 μg/mL with cell adherence disappear and all cells apoptosis.The Ct value of INS-1-SEMS2 cells detected at the second time was 28.21, which was greater than 27.58 at the first time.The interfering efficiency of siRNA was 77.78% which indicated a successful expression of siRNA and construction of monoclonal stable cell line ( INS-1-SEMS2 ).Conclusion The monoclonal stable cell line was successfully constructed by lentivirus vector-mediated RNA interence silenced gene SGMS2.