Molecular Characterization Of A Human Matrix Attachment Region That Improves Transgene Expression In CHO Cells

BackgroundTherapeutic recombinant proteins can be produced using genetic engineering technology.Currently most of therapeutic recombinant proteins are produced by Chinese hamster ovary(CHO)cell expression system.However,instability expression and low expression level of CHO cell lines are important factors which restrict the application of CHO cells.Previous studies found that matrix attachment region(MAR)can significantly increase transgene expression level,buffer transgene silencing.However,the mechanism and the key elements of matrix attachment regions regulate the transgene expression have been unclear.ObjectiveThe aim of this study was to analyze the molecular characteristics and identify the key elements of MAR that can improve transgene expression in stably transfected CHO cells.MethodsThe six sub-fragments from 5'-3'(1-540,420-1020,900-1500,1380-1980,1860-2460 & 2340-2999)from flanking regions of the ?-globin MAR were amplified using PCR technology.The adjacent sub-fragments were all overlapped 120 bp.Bidirectional cloning was achieved by digesting the PCR amplified fragments with Kpn I and Bgl II,and subsequently inserting them in pCATG plasmid digested with the above same restriction enzymes to generate the six plasmids,named pCAM1-6,respectively.Cells were divided into the following eight groups: pCAM1-6;pCAM and pCATG as control group.Followed,the stably transfected CHO cells were cultured under G418,and chloramphenicol acetyltransferase(CAT)content was analyzed using ELISA method.The characteristics of the MAR sequence were analyzed using bioinformatics.ResultsThe results showed that the overall length of ?-globin MAR can significantly improve the expression level of CAT.The second and third sub-fragments(position 421-1 020,901-1 500)showed the most effective function compared to other sub-fragments.The bioinformatics analysis results showed that MAR-2 contains one MAR-like motif(AATATATTT);MAR-3 contains one MAR-like motif(AATATATTT)and two Topo II site;MAR-6 contains one Alu conservative sequence and ?-globin cluster.ConclusionMAR element can improve the transgenic expression in mammalian cells.The bioinformatics analysis results showed that MAR-like motif of MAR may contribute to transgene expression increasing.