title: Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells.
abstract: Chinese hamster ovary (CHO) cells offer many advantages for recombinant gene expression, including proper folding and post-translational modification of the recombinant protein. However, due to positional effects resulting from the neighboring chromatin, transgenes are often expressed at low levels in these cells. While previous studies demonstrated that matrix attachment regions (MARs) can be utilized to increase transgene expression by buffering transgene silencing, the mechanism by which this occurs is poorly understood. We therefore performed a deletion analysis of the human β-globin MAR sequence to characterize the regions that are necessary to enhance transgene expression in CHO cells. Our results indicate that of the six β-globin MAR fragments tested (MAR-1-6; nucleotides 1-540, 420-1020, 900-1500, 1380-1980, 1860-2460, and 2340-2999, respectively), MAR-2, followed by MAR-3, was the most effective region for promoting stable and elevated transgene expression. Meanwhile, bioinformatic analyses demonstrated that these fragments encode a MAR-like motif and several transcription factor binding sites, including special AT-rich binding protein 1 (SATB1), CCAAT-enhancer-binding proteins (C/EBP), CCCTC-binding factor (CTCF), and Glutathione (GSH) binding motifs, indicating that these elements may contribute to the MAR-mediated enhancement of transgene expression. In addition, we found that truncated MAR derivatives yield more stable transgene expression levels than transgenes lacking the MAR. We concluded that the MAR-mediated transcriptional activation of transgenes requires a specific AT-rich sequence, as well as specific transcription factor-binding motifs.
authors: Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan 453003, China;
authors: Qiu-Li
authors: QL
authors: Sun
authors: Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan 453003, China;
authors: Chun-Peng
authors: CP
authors: Zhao
authors: Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan 453003, China;
authors: Shao-Nan
authors: SN
authors: Chen
authors: Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan 453003, China;
authors: Li
authors: L
authors: Wang
authors: Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan 453003, China;; Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, Xinxiang, 453003, Henan, China. Electronic address: wtianyuncn@126.com.
authors: Tian-Yun
authors: TY
authors: Wang
mesh_terms: D000818:Animals; D016466:CHO Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D057075:Enzyme Assays; D015870:Gene Expression; D006801:Humans; D045170:Matrix Attachment Regions / Q000235:genetics*; D011485:Protein Binding; D017384:Sequence Deletion; D014157:Transcription Factors; D019076:Transgenes; D055544:beta-Globins / Q000235:genetics
chemical_list: D014157:Transcription Factors; D055544:beta-Globins; D015500:Chloramphenicol O-Acetyltransferase
keywords: Chinese hamster ovary; Gene expression; Matrix attachment region; Molecular characterization; Transgene silencing