MAR Combinations Increase Transgene Expression In Recombinant Chinese Hamster Ovary Cells

BackgroundMammalian cell expression systems are widely used to express recombinant proteins for clinical applications owing to their capacity for post-translational modification and assembly of human protein-like molecular structures.Chinese hamster ovary(CHO)cells have been the preferred mammalian cell line for production of recombinant therapeutic applications proteins.Transgene silencing and low expression levels are common problems in recombinant protein production.We previously demonstrated that MARs can enhance CHO transgene expression.However,the mechanism and function of MAR combinations on transgene expression are unclear.ObjectiveThe aim of this study was to investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms.MethodsThe human ?-interferon MAR(iMAR)and ?-globin MAR(gMAR)were amplified through PCR using the primers designed according to the GenBank sequences.Using seamless cloning technique,we generated constructs in which the enhanced green fluorescent protein(EGFP)gene flanked by different combinations of i MAR and gMAR,respectively,which were driven by the cytomegalovirus(CMV)or simian virus(SV)40promoter.Following the vectors were transfected into CHO-K1 cells,transfection efficiency and transient EGFP expression were analyzed by fluorescence microscope;after48 h,the transfected cell were screened with geneticin,and the stably tranfected cell were screened,EGFP expression was detected by flow cytometry.Bioinformatics was furtherused to analyze the effect and mechanism of MAR combination on transgenic expression of recombinant CHO cells.ResultsThe presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters;the level was higher when the two MARs differed(i.e.,iMAR and gMAR)under the CMV but not the SV40 promoter.For the latter,two gMARs showed the highest activity.We also found that MARs increased the ratio of stably transfected positive colonies.ConclusionCombining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.