Weakening resistance marker for establishing a process of screening high-producing CHO cell lines
10.11665/j.issn.1000-5048.20150517
- VernacularTitle:弱化抗性标记筛选高表达 CHO 细胞株的方法建立
- Author:
Su LIU
;
Hong TIAN
;
Chi WANG
;
Wenbing YAO
- Publication Type:Journal Article
- Keywords:
Chinese hamster ovary cell;
neomycin-phosphotransferase;
enhanced green fluorescent protein;
high expression system
- From:
Journal of China Pharmaceutical University
2015;(5):617-622
- CountryChina
- Language:Chinese
-
Abstract:
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 bear-ing 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.
