CHO-DG44细胞瞬时转染条件的优化

Optimization of condition for transient transfection of CHO-DG44 cells

目的优化CHO-DG44细胞瞬时转染的条件。方法采用TubeSpin一次性生物反应器,以绿色荧光蛋白(Green fluorescence protein,GFP)为报告基因,氯醚酰亚胺(Polyether imide,PEI)为转染试剂,将质粒pIRESneo3-eGFP瞬时转染CHO-DG44细胞,优化瞬时转染的基本条件[细胞密度、DNA浓度和DNA:PEI比例(w/w)]以及其他条件(换液、渗透压和温度),采用优化的条件转染质粒pIRESneo3-eGFP,流式细胞术检测细胞转染效率,生物发光仪检测相对荧光强度。结果 CHO-DG44细胞瞬时转染的最佳条件为:采用XLG-P8培养基进行转染,细胞密度为2×106个/ml,DNA浓度为6.25μg/5 ml,DNA∶PEI(w/w)比例为1∶5;转染后4 h更换新鲜培养基,添加30 mmol/L NaCl,并于31℃继续培养。在此条件下,CHO-DG44细胞的瞬时转染效率可达81.45%,相对荧光强度可达9×105RFU/106cells。结论优化了CHO-DG44细胞瞬时转染的条件,为下一步药物蛋白的研发奠定了基础。

Objective To optimize the condition for transient transfection of CHO-DG44 cells.Methods CHO-DG44 cells were transiently transfected with plasmid pIRESneo3-eGFP in TubeSpin disposable bioreactor using green fluorescence protein（GFP） gene as a reporter and polyether imide（PEI） as a transfection reagent.The primary condition such as cell density,DNA concentration and ratio of DNA to PEI（w / w）,as well as other conditions such as osmotic pressure and temperature for changing medium,were optimized.The cells were transfected with pIRESneo3-eGFP under the optimized condition,and determined for transfection efficacy by flow cytometry,and for relative fluorescent intensity by bioluminescence analyzer.Results The optimal medium,cell density,DNA concentration,ratio of DNA to PEI（w / w） for transient transfection of CHO-DG44 cells were XLG-P8 medium,2 × 10^6 cells / ml,6.25 μg / 5 ml and 1： 5 respectively.The culture was transferred to fresh medium 4 h after transfection,added with 30 mmol / L sodium chloride and further incubated at 31℃.Under the optimized condition,the transient transfection efficiency of CHO-DG44 cells reached 81.45%,while the fluorescent intensity was about 9 × 10^5 RFU / 106 cells.Conclusion The condition for transient transfection of CHO-DG44 cells was optimized,which laid a foundation of further development of pharmaceutical proteins.