A “GC-rich” method for mammalian gene expression: A dominant role of non-coding DNA GC content in regulation of mammalian gene expression

Abstract

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein. The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment (including intron) of extremely GC-rich at the 5' or/and 3' flanking regions of these protein genes or their gene promoters. This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super "chromatin opening element" and plays an important role in mammalian gene expression. This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure, namely DNA GC-content.

The chimeric monoclonal antibody MHCSZ-123 against human von Willebrand factor A3 domain inhibits high-shear arterial thrombosis in a Rhesus monkey model

“…To produce chimeric monoclonal antibody MHCSZ-123 effectively, we chose pMH3, which is an expression vector that contains ubiquitous chromatin opening elements (UCOEs), transfected into CHO-S cells as an expression system [26]. Detailed structures of pMH3-MHC-SZ-123VH and pMH3-MHC-SZ-123VL expression vectors are depicted in Fig.…”

“…The pMH3 vector (AmProtein, Hangzhou, China), a eukaryotic expression vector that includes the chicken beta actin gene intron-1 promoter (GC content average 75.3%, the highest 90.8%), was selected for high expression of the chimeric antibody [26]. pMH3H and pMH3L vectors carried the cDNAs of the IgG1 heavy chain and kappa chain constant regions, respectively, which resulted in GC-rich DNA structures.…”

Efficient production of FAM19A4, a novel potential cytokine, in a stable optimized CHO-S cell system

“…To produce recombinant FAM19A4 protein effectively, we chose pMH3, an UCOE containing expression vector, in CHO-S expression system [15]. Detailed structure of pMH3-FAM19A4 expression vector was depicted in Fig.…”

“…For efficient production of recombinant protein, the expression vector is essential in addition to the host cell. pMH3 is an eukaryotic expression vector marking ''GC-rich'' that is suitable for CHO expression system [15]. The vector is designed and modified for increasing transcriptional activity of exogenous genes.…”

“…It consists of three non-coding GC-rich DNA regions which can regulate chromatin openness and are novel UCOEs. Therefore, the UCOE containing vector pMH3 can protect the interesting molecules from silencing, which leads to high efficiency and stable production of proteins no matter where their integration sites are [15,16].…”