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

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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 / m L,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 titer: 1 × 108 TU / m L) 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 / m L 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.

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