流式细胞术结合G418筛选重组质粒稳定转染细胞

目的:探索优化重组质粒转染细胞后的筛选方法,以得到高纯度U937转染细胞。方法:选择带Neor及GFP基因的质粒pRNATU6.1/Neo。根据流式细胞仪可分选GFP阳性细胞这一特点,在质粒转染细胞后比较单用G418筛选与合并使用流式细胞仪分选后的筛选效果。结果:单用G418筛选时最高筛选效率为72.0%;合并使用G418及流式细胞仪分选后的筛选效率均在90.0%以上。结论:合并使用G418及流式细胞仪分选是一种高效的筛选方法。

Combined use of flow cytometry and G418 to screen the recombinant plasmid transfected cells

Objective To optimize the selective method to obtain the pure recombinant plasmid transfected U937 cells. Methods pRNAT U6.1/Neo plasmid containing both Neor and GEP genes was selected. After the plasmids had been transfected into U937 cells, the purity of the transfected cells, selected by G418 only or by both G418 and flow cytometer (FCM), was compared. Results The highest purity of GFP positive cells was 72.0% when selected by G418 only; however, the purity of GFP positive cells was always above 90.0% while using both G418 and FCM method. Conclusions It is a highly effective method to select the transfected cells using both G418 and FCM.