The Effect Of Human DNA Topoisomerase I (TOPI) MAR On Transgenic Expression In CHO Cells

Background Stable and efficient transgene expression is required for the industrial production of therapeutic proteins by using mammalian cell lines,especially Chinese hamster ovary(CHO)cells.Matrix attachment region(MAR),as a sort of DNA cis-acting element containing different characteristic motifs,is favourable for overcoming gene silencing and enhancing transgenic expression.However,the effect of MAR which has been reported on enhancement of expression need to be improved,and the mechanism of its function has not yet clear.Objective To investigate the effect of a new humanized TOPI(human DNA topoisomerase I gene)MAR sequence on transgene expression and stability in CHO cells and to explore the factors affecting expression.Methods3.Vector construction and cell transfection: The TOPI sequence was cloned by PCR amplification.Three expression vectors containing TOPI MAR sequence were constructed to expression target gene: enhanced green fluorescence protein(EGFP).TOPI MAR sequences located at the upstream,downstream and flank of EGFP expression cassette,respectively.The constructed expression vectors were transfected into CHO-S cells.4.Transient expression analysis of target gene: After CHO cells transfected for 48 hours,the transient expression level of EGFP was observed by fluorescence microscope and quantified by flow cytometry.5.Stable expression analysis of the target gene: The polyclonal cell pools were obtained y G418 screening,and then were cultured for 30 generations in culture medium with or without G418 screening,respectively.The expression level of EGFP was detected by flow cytometry.The monoclonal CHO recombinant cell lines were screened by stepwise dilution method and the expression level of EGFP was detected by flow cytometry.The copy number and m RNA expression of target gene EGFP in CHO cells on molecular level were tested by quantitative real-time PCR(q PCR).6.The expression of recombinant protein erythropoietin(EPO): The EPO gene was cloned by PCR amplification.The expression vectors containing TOPI MAR sequence and target protein EPO were constructed and transfected into CHO cells.The protein expression level of EPO in recombinant CHO cells were detected by Western blot.Results1.The TOPI MAR sequence was successfully constructed.The eukaryotic expression vectors containing TOPI MAR sequence used for expression target genes EGFP and EPO were successfully constructed.2.The results of flow cytometry showed that TOPI MAR sequence inserted in different positions can improve the expression levels of target gene EGFP.The expression reached higher level when TOPI MAR sequence located at the flank and downstream of the EGFP expression cassette.3.The results of long-term expression stability indicated that the maintenance rate of transgenic expression was the highest when the TOPI MAR sequence located at the downstream of the expression cassette.4.The results of q PCR demonstrated that the transgene expression level could be improved by increasing the copy number of target gene and expression of m RNA.5.The results of Western blot revealed that TOPI MAR sequence significantly improved the expression level of EPO when the TOPI MAR sequence was inserted at downstream of the expression cassette.Conclusion1.The expression vector containing TOPI MAR sequence can improve transgene expression level and stability in recombinant CHO cells2.The copy number and the expression level of m RNA of target gene as well as the position effect of TOPI insertion are the main factors that affect the improvement of TOPI MAR on transgene expression.