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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baldi A, De Luca A, Morini M et al (2002) The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21:6684–6688
Chang YN, Crawford S, Stall J et al (1990) The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements. J Virol 64:264–277
Chang YN, Jeang KT, Chiou CJ et al (1993) Identification of a large bent DNA domain and binding sites for serum response factor adjacent to the NFI repeat cluster and enhancer region in the major IE94 promoter from simian cytomegalovirus. J Virol 67:516–529
Chien J, Staub J, Hu SI et al (2004) A candidate tumor suppressor HtrA1 is downregulated in ovarian cancer. Oncogene 23:1636–1644
Chien J, Aletti G, Baldi A et al (2006) Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity. J Clin Invest 116:1994–2004
Elateri I, Muller-Weeks S, Caradonna S (2003) The transcription factor, NFI/CTF plays a positive regulatory role in expression of the hSMUG1 gene. DNA Repair 2:1371–1385
Foecking MK, Hofstetter H (1986) Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 45:101–105
Hou S, Maccarana M, Min TH et al (2007) The secreted serine protease xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo. Dev Cell 13:226–241
Iwai R, Kumagai Y, Fujiwara M et al (2010) Combination of cytomegalovirus enhancer with human cellular promoters for gene-induced chondrogenesis of human bone marrow mesenchymal stem cells. J Biosci Bioeng 110:593–596
Jeang KT, Rawlins DR, Rosenfeld PJ et al (1987) Multiple tandemly repeated binding sites for cellular nuclear factor 1 that surround the major immediate-early promoters of simian and human cytomegalovirus. J Virol 61:1559–1570
Kiefer K, Clement J, Garidel P et al (2004) Transfection efficiency and cytotoxicity of nonviral gene transfer reagents in human smooth muscle and endothelial cells. Pharm Res 21:1009–1017
Koster RW, Fraser SE (2001) Tracing transgene expression in living zebrafish embryos. Dev Biol 233:329–346
Liu BH, Wang X, Ma YX et al (2004) CMV enhancer/human PDGF-beta promoter for neuron-specific transgene expression. Gene Ther 11:52–60
Mehta AK, Majumdar SS, Alam P et al (2009) Epigenetic regulation of cytomegalovirus major immediate-early promoter activity in transgenic mice. Gene 428:20–24
Nüsslein-Volhard C, Dahm R (2002) Zebrafish: a practical approach, 1st edn. Oxford University Press, Oxford
Suhr ST, Ramachandran R, Fuller CL et al (2009) Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock. Gene Expr Patterns 9:54–64
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).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-011-0589-5