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

Abstract:

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.