Abstract
Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR‐3 and MAR‐7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real‐time PCR. The results showed that the expression level of eGFP of cells transfected with MAR‐containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR‐7 was higher than that of MAR‐3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR‐3 and MAR‐7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.
Highlights
Mammalian cell expression systems are generally the preferred platform for producing the recombinant therapy proteins, as they can generate the large and complex proteins with post‐translational modifications similar to those produced in humans.[1,2] More than 70% of the recombinant therapeutic proteins were produced by the Chinese hamster ovaries (CHO) cells.[3]
The results showed that the mean fluorescence intensity (MFI) of enhanced green fluorescent protein (eGFP) containing Matrix attachment regions (MARs)‐3 and MAR‐7 was 2.79‐ and 2.92‐fold compared with the control vector respectively (Figure 1B), suggesting two MAR elements can increase the transgene expression level of stably transfected cells
We investigated the effects of MAR‐3 and MAR‐7 on transgene expression in CHO cells using eGFP reporter gene
Summary
Mammalian cell expression systems are generally the preferred platform for producing the recombinant therapy proteins, as they can generate the large and complex proteins with post‐translational modifications similar to those produced in humans.[1,2] More than 70% of the recombinant therapeutic proteins were produced by the Chinese hamster ovaries (CHO) cells.[3]. MARs are widely used to improve the high level of expression of transgenic genes and to prevent epigenetic silencing by blocking the transmission of heterochromatin.[6,7] Up to now, it has been reported that several MAR elements can increase the level of transgene expression as well as the proportion of positive colonies in CHO cell expression systems, and reduce the differences of expression between different transformed strains.[8,9,10] We identified two new, more powerful MAR elements from the human genome DNA, which can be used to improve the transgene expression in transfected CHO cells
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