| BackgroundRecombinant therapeutic proteins have became an important part of biomedicine industry.Mammalian expression system is the main platform for the production of recombinant therapeutic proteins,and the Chinese hamster ovarian cell(CHO)expression system is the most widely used mammalian expression system.However,there are some issues in CHO cell expression system,such as transgene silencing or unstable expression level.It was found that as an epigenetic regulatory element,nuclear matrix attachment regions(MAR)can not only overcome the silencing of transgenic genes,but also improve the expression level of transgenic genes.However,the reported MAR sequences used to improve transgene expression are limited,and the mechanism and molecular characteristics of its regulation of transgene expression are still unclear.ObjectiveThe aim of this study was to screen novel MARs which can effectively improve the level of transgene expression in CHO cells and to analyze its mechanism and molecular characteristics.Methods 1.Vector construction and cell transfection: Human CSP-B MAR(hu-MAR in this paper),MAR-6(MAR1 in this paper)and DHFR intron MAR(MAR2 in this paper).Different MAR sequences were cloned into downstream of expression vector containing enhanced green fluorescent protein(eGFP),respectively.The constructed vector was transfected into CHO-S cells.2.Transient expression analysis: The transient expression of eGFP reporter gene was observed by fluorescence microscope and the transfection efficiency of different vectors was analyzed after 48 h of transfection.3.Stable expression analysis: Geneticin(G418)was added into the cell pool to screen and obtain polyclonal stable cell pool(cell pool)after 48 h of transfection.When the cells were screened under pressure for 20 generations,the expression level of reporter gene eGFP was analyzed by flow cytometry.4.Gene copy number analysis: Real-time fluorescence quantitative PCR(qPCR)was used to detect the copy number of target gene eGFP.5.Stability analysis of long-term expression: The cell pools were cultured for 40 generations in culture medium with G418.The expression level of reporter gene eGFP was analyzed by flow cytometry.6.Expression of bevacizumab:,The heavy and light chain genes of bevacizumab replaced the reporter gene eGFP of the above vectors.Then the vectors were transfected into CHO-S cells and G418 was added into culture medium to screen and obtain the stable cell pools.The expression levels of bevacizumab in CHO-S cells were detected by western blot.Results 1.The resultant eukaryotic expression vectors containing three different MAR sequences and the reporter gene eGFP and the bevacizumab were successfully constructed.2.The transient expression of hu-MAR,MAR2,MAR1 showed that the transfection efficiency and the transient expression level of eGFP were increased by 96%,89%,83% and 2.3 fold,2.0 fold and 1.5 fold,respectively.3.The results showed that hu-MAR,MAR2,MAR1 could increase the stable expression level of eGFP by 4.5 fold,2.7 fold and 2.5 fold,respectively.4.The analysis of gene copy number showed that there was no significant correlation between the eGFP expression levels and gene copy.hu-MAR,MAR2,MAR1 relative copy number of eGFP gene were 2.80 ±0.43,6.40 ±0.89 and 6.90 ±1.21,respectively.5.The results of long-term expression stability analysis showed that hu-MAR,MAR2,MAR1 could maintain the stable expression level of eGFP,and the maintenance rates were 70%,65% and 43%,respectively.6.The results showed that hu-MAR,MAR2,MAR1 increased the expression level of bevacizumab by 1.46 fold,1.09 fold and 1.15 fold,respectively.Conclusion 1.hu-MAR,MAR2,MAR1 could improve the level and stability of transgene expression,among which the hu-MAR showed the most significant effect on transgenic expression.2.Two kinds of transcription factor binding sites,NFAT and VTBP,possibly contribute to the improvement of transgene expression. |
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