Weakening resistance marker for establishing a process of screening high-producing CHO cell lines

To optimize Chinese hamster ovary(CHO)expression system and establish a process of screening CHO cell lines with high productivity, neomycin-phosphotransferase(NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G. After selection by culturing with G418, the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bearing wide type NPT. An enhanced green fluorescent protein(EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene, which verified that the resistance of mutant-NPT to G418 was weakened. By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks, mutant-selected pools expressed more exogenous protein than the WT-selected pools. Therefore, the ratio of high producers in a transfected cell population greatly increased.