CHO细胞Kcmf1基因内定点整合ZsGreen1报告基因的表达稳定性研究

Expression Stability of ZsGreen1 Reporter Gene in Kcmf1 Gene of CHO Cells

以Kcmf1基因NW_003614172.1第629890碱基处定点整合ZsGreen1报告基因的CHO-K1细胞(2C3)为研究材料,采用倒置荧光显微镜和流式细胞仪定量分析ZsGreen1的表达,开展系统的稳定性表达研究。2C3细胞贴壁培养连续传代50代次,100%的细胞可以稳定表达ZsGreen1报告基因。无血清悬浮驯化培养2C3细胞后,细胞表达ZsGreen1的平均荧光强度明显降低;连续悬浮传代培养60代次,表达ZsGreen1的细胞数目显著增多,添加体积分数10%FBS到悬浮培养的细胞池,ZsGreen1阳性细胞比例上升到95%以上。流式细胞术分选细胞池中高、低表达ZsGreen1蛋白的2C3细胞,PCR确认它们均含有ZsGreen1报告基因;Q-PCR发现在高、低表达ZsGreen1蛋白的细胞中,相关的ZsGreen1 mRNA水平有明显的差异。提示CHO-K1细胞Kcmf1基因内非编码区域定点整合外源基因后,不会因细胞分裂和生长而丢失外源表达基因;悬浮驯化需要优化培养基和培育条件,达到构建工程细胞的需求。

The expression of ZsGreen1 in CHO-K1 cells(2C3)with the ZsGreen1 reporter gene integrated at the 629890 base of Kcmf1 gene NW_003614172.1 was quantitatively analyzed by inverted fluorescence microscope and flow cytometry to carry out the systematic stability expression study.The ZsGreen1 reporter gene could be expressed stably in 100%of 2C3 cells after continuous passage of 50 generations in adherent culture.After cultured without serum,the average fluorescence intensity of ZsGreen1 expression in 2C3 cells decreased significantly.After 60 generations of continuous suspension culture,the number of cells expressing ZsGreen1 increased significantly.When 10%FBS was added to the cell pool of suspension culture,the proportion of ZsGreen1 positive cells increased to over 95%.Flow cytometry sorted 2C3 cells with high and low expression of ZsGreen1 protein in the cell pool,and PCR confirmed that they all contained the ZsGreen1 reporter gene.Q-PCR found significant differences in the levels of related ZsGreen1 mRNA in cells with high and low expression of ZsGreen1 protein.It was suggested that the non-coding region of Kcmf1 gene in CHO-K1 cells integrated exogenous genes at specific sites,and the exogenous expressed genes would not be lost due to cell division and growth.The suspension and acclimation should optimize the culture medium and cultivation conditions to meet the needs of engineering cells construction.