| Chinese hamster ovary?CHO?cells have been the most widely used production cells for biotechnology drugs.The focus of research work is to construct CHO engineering cell lines suitable for the stable production of therapeutic biological products.In the early stage,the research group used CHO-K1 as the starting cell and combined the lentiviral tracing reporter gene technology and chromosome walking technology to find 6 intragenomic sites that can continuously express the reporter gene.In this study,the expression stability of one of the sites was verified,the site-specific integration and stable expression of enhanced green fluorescent protein?EGFP?gene and human serum albumin?HSA?genes were achieved at this site,respectively.?.Using the CHO-K1-ZsGreen1-2C6 cells with the ZsGreen1 reporter gene integrated as the research material,the expression of the ZsGreen1 reporter gene was quantitatively analyzed by using an inverted fluorescence microscope and flow cytometry.According to the cell generation requirements of the main cell bank to the 2000L cell production tank scale-up culture,continuous subculture for 50 generations,it was found that 100%of CHO-K1-ZsGreen1-2C6 adherent cells and suspended cells can stably express the ZsGreen1 reporter gene,suggesting that CHO-K1 cell LOC103162981 gene NW?003626341.1 in the 1689 base?upstream and downstream 1614?1763base range?can be used as an integration site for the stable expression of exogenous genes.?.Based on CRISPR/Cas9 gene editing technology and homologous targeted repair pathway,a donor fragment containing a dual selection tag of puromycin and fluorescent protein was designed.Using CHO-K1 cells and BAK-/BAX--CHO-K1 cells which apoptosis-related genes BAK and BAX were double knocked out as the starting cell lines,EGFP reporter gene and HSA gene were successfully knocked in the genome NW?003626341.1 at the 1642 base?47 bp upstream of the1689th base?.A total of nine CHO-K1-EGFP monoclonal cell lines,eleven BAK-/BAX--CHO-K1-EGFP monoclonal cell lines,five CHO-K1-HSA monoclonal cell lines and four BAK-/BAX--CHO-K1-HSA monoclonal cell lines were obtained by PCR amplification.Statistical analysis indicated that 19%to 23%of the monoclonal cell lines accurately knocked in the EGFP gene,and8.5%to 12%accurately knocked in the HSA gene.?.Adapting the adherent cells knocked into the target gene to suspension culture,the expression of EGFP gene in CHO-K1-EGFP cells and BAK-/BAX--CHO-K1-EGFP cells was analyzed by flow cytometry.It was found that during 50 generations of continuous suspension subculture,100%of the EGFP reporter gene knock-in cells can stably express the EGFP protein,and the unit fluorescence intensity was not lower than that of the original cells,which verified the stability of the expression of exogenous genes at this integration site.The HSA gene knock-in cells were cultured in batches,and the expression level of HSA protein was quantitatively detected.Compared with the starting cell line,there was no significant change in the growth status of knock-in cells.Two strains of CHO-K1-HSA cells with high protein expression were obtained:CHO-K1-HSA-7 and CHO-K1-HSA-50 cells.After 8 days of batch culture,the protein accum-ulation was 228.50 mg×L-1 and 219.25 mg×L-1.One strain of BAK-/BAX--CHO-K1-HSA cells with high protein expression was obtained:BAK-/BAX--CHO-K1-HSA-9 cells,the protein accumulation amount reached 97.24 mg×L-1 on the 9th day of batch culture.Through stability verification and expression level analysis,it was determined that the 1642 base in NW?003626341.1 of the LOC103162981 gene could be used as an integration site to become a reliable choice for researchers to construct CHO engineering cell lines that stably express therapeutic proteins. |
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