Abstract
Human and simian cytomegalovirus immediate-early (IE) enhancer/promoter (hCMVp and sCMVp) are the most widely used system for high-level gene expression; however, studies on detailed comparative analyses of the promoters are scarce. Using GFP reporter gene and immunoblotting assays, we have shown that the transcriptional activity of sCMVp was two to four fold higher than those of hCMVp in human-, monkey-, mouse-originated cell lines, and zebrafish as a vertebrate animal model. Notably, HtrA1 driven by the sCMVp induced cell death at relatively high-levels in HEK293 cells, but HtrA1 driven by the hCMVp had almost no effect on cell death, as shown by more than 4-fold increase in the expression levels of HtrA1. Our data may provide a valuable tool for functional studies of target genes that are expressed at extreme low level under standard transfection conditions and for development of new gene therapeutic systems.
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Acknowledgments
We thank Dr. Alfonso Baldi (Second University of Naples, Italy) for the pcDNA-T7-PRSS11 (HtrA1) plasmid, Drs David Turner and Ralph Rupp (University of Michigan, Ann Arbor, MI) for the pCS2+ plasmid, and Cheol-Hee Kim (Chungnam National University, Korea) for the pCS2+ EGFP plasmid. This study was supported by the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs (A080418).
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10529_2011_589_MOESM1_ESM.tif
Supplementary Fig. 1. Sequence alignment of the 5′-upstream enhancer and promoter regions in the sCMVp and hCMVp. The sequence alignment was performed by ClustalW program. Promoter region (open box). Both CMVp regions include different sets of repetitive elements, such as serum response transcriptional factor (SRF)-binding sites (SREs, red box) and of cAMP response elements (CREs, yellow box). The sCMVp contains additional cellular nuclear factor I binding sites (NEs, green box). (TIFF 16986 kb)
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Kim, GY., Moon, JM., Han, JH. et al. The sCMV IE enhancer/promoter system for high-level expression and efficient functional studies of target genes in mammalian cells and zebrafish. Biotechnol Lett 33, 1319–1326 (2011). https://doi.org/10.1007/s10529-011-0589-5
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DOI: https://doi.org/10.1007/s10529-011-0589-5