A comparative study on transfection efficiency of green fluorescent protein after concentration of lentivirus by three methods
BI Ranran1,BAI Rui1,LIU Zhiqiang2,and LIU Huiliang3
1.Department of Internal Medicine-Cardiovascular,3.Institute of Cardiology,the Third Medical Centre,Chinese PLA General Hospital,Beijing 100039,China; 2.Institute of Military Cognition and Brain Science,PLA Military Medical Research Institute,Beijing 100850,China
Abstract：Objective To compare the transfection efficiency of green fluorescent protein (GFP) after concentration of lentivirus by ultrafiltration tube, ultracentrifugation and virusconcentrate. Methods pMD2.G, psPAX2 and GFP vector strains were shaken overnight in a shaker and the three plasmids were extracted with kits.pMD2.G, psPAX2 and GFP vector plasmids were proportionally mixed with transfection reagents and transfected into 293T cells to produce virus.The virus supernatant was collected and concentrated by ultrafiltration tube, ultracentrifugation and virus concentrate. The 293T cells were inoculated into three groups, and the concentrated viral concentrates of 2, 4, 8,16 μl were added into the culture medium of 293T cells inoculated into each group, after 48 hours, the expression of GFP in 293T cells transfected with lentivirus was detected by fluorescence microscopy and flow cytometry. Results The results of fluorescence microscopy showed that the expression rate of GFP increased with the increase of the transfection amount, and the highest expression rate of GFP was obtained when the virus was transfected into 293T cells by ultrafiltration tube. Flow cytometry showed that the expression rates of 2, 4, 8,16 μl GFP in virus concentrate group were (2.4±0.1)%,(3.8±0.3)%,(8.2±0.6)% and(12.2±0.3)% respectively; the expression rates of 2, 4, 8,16 μl GFP in ultrafiltration tube group were (5.4±0.3)%,(7.4±0.4)%,(12.7±0.1)% and (17.3±0.6)% respectively; the expression rates of 2, 4, 8,16 μl GFP in ultracentrifugation group were (0.7±0.1)%,(1.2±0.1)%,(2.1±0.1)% and (0.4±0.2)%, respectively. Compared with the other two groups, the ultrafiltration tube group had significant difference(P<0.05). Conclusion The transfection efficiency of lentiviral GFP is different by different methods. The transfection efficiency of lentiviral GFP by ultrafiltration tube is the highest among the three lentiviral enrichment methods.
毕冉冉, 白睿, 刘志强, 刘惠亮. 三种方法浓缩慢病毒后绿色荧光蛋白转染效率的比较[J]. 武警医学, 2019, 30(1): 62-66.
BI Ranran,BAI Rui,LIU Zhiqiang,and LIU Huiliang. A comparative study on transfection efficiency of green fluorescent protein after concentration of lentivirus by three methods. Med. J. Chin. Peop. Armed Poli. Forc., 2019, 30(1): 62-66.
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2019, Vol. 30 
