Enhanced transgene expression using two β-globin MARs flanking expression cassettes in stably transfected CHO-K1 cells
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In this study, we systemically investigated the positions and orientations of matrix attachment regions (MARs) in expression vectors to fully explore the mechanism for improving transgene expression. We constructed 14 vectors that incorporated human β-globin MARs into pIRES-eGFP backbone vectors. The MARs flanked the eGFP expression cassette or promoter in a forward/reverse orientation. After stable transfection into CHO-K1 cells with these vectors, eGFP expression levels were increased significantly relative to that of the control vector (MAR-devoid) when two MARs flanking the expression cassette were incorporated, followed by those at the 5′ site (upstream of the promoter). Simultaneously, the percentage of the eGFP-expressing cells was elevated to some extent. The vector with both MARs in forward orientation flanking the expression cassette yielded the highest transgene expression levels (2.5-fold). The orientation (forward or reverse) of the MARs did not present a significant difference when added in the same site. In addition, transgene expression levels were not exclusively dependent on transgene copy numbers. Bioinformatic analysis indicated that some specific transcription factors may contribute to the transcriptional process. In conclusion, two MARs in a forward orientation and flanking the expression cassette comprised the optimal construct for improving the stable transgene expression in the CHO-K1 cells. The effects may be related to specific transcription factors, such as PRDM1 and REL.
KeywordsMatrix attachment region Position effect Transgene expression Transcription factor-binding site
This work was supported by the Grant from the National Natural Science Foundation of China (no. U1604193).
JZ designed, analyzed the experiments and wrote the manuscript. JZ performed the experiments and co-wrote the manuscript. SC and WY analyzed some experiment data and co-wrote the manuscript. SL performed the experiment for vector construction and cultured cells.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
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