Abstract
The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci USA 102(36):12678–12683
Bucher P (1990) Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. J Mol Biol 212(4):563–578
Chung JH, Bell AC, Felsenfeld G (1997) Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci USA 94(2):575–580
Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ, Lowe SW (2005) Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37(11):1289–1295
Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD (2001) Analysis of the aphthovirus 2A/1a polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol 82(Pt 5):1013–1025
Ferreira JP, Lawhorn IEB, Peacock RWS, Wang CL (2011) Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters. Int Biol. doi:10.1039/C1IB00082A
Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP, Lin-Cereghino J, Kovar K, Cregg JM, Glieder A (2008) Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 36(12):e76
Jensen PR, Hammer K (1998a) Artificial promoters for metabolic optimization. Biotechnol Bioeng 58(2–3):191–195
Jensen PR, Hammer K (1998b) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 64(1):82–87
Matsuyama A, Yoshida M (2009) Systematic cloning of an ORFeome using the Gateway system. Methods Mol Biol 577:11–24
Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol 70(8):5701–5705
Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, Stephanopoulos G (2006) Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol 72(8):5266–5273
Rud I, Jensen PR, Naterstad K, Axelsson L (2006) A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiology 152(Pt 4):1011–1019
Ruth C, Glieder A (2010) Perspectives on synthetic promoters for biocatalysis and biotransformation. ChemBiochem 11(6):761–765
Sarkisian CJ, Keister BA, Stairs DB, Boxer RB, Moody SE, Chodosh LA (2007) Dose-dependent oncogene-induced senescence in vivo and its evasion during mammary tumorigenesis. Nat Cell Biol 9(5):493–505
Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88(5):593–602
Thomsen DR, Stenberg RM, Goins WF, Stinski MF (1984) Promoter-regulatory region of the major immediate early gene of human cytomegalovirus. Proc Natl Acad Sci USA 81(3):659–663
Tornoe J, Kusk P, Johansen TE, Jensen PR (2002) Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297(1–2):21–32
Xi H, Yu Y, Fu Y, Foley J, Halees A, Weng Z (2007) Analysis of overrepresented motifs in human core promoters reveals dual regulatory roles of YY1. Genome Res 17(6):798–806
Acknowledgments
We thank Scott Lowe (Cold Spring Harbor Laboratory, USA) for vector reagents. We thank Kathyrn Sullivan for help in engineering promoters and Wes Overton and Goutam Nistala (all three from Stanford University, USA) for proofreading the manuscript. This work was supported by the NSF (CAREER 0846392), Ellison Medical Foundation (New Scholar in Aging AG-NS-0550-09), and Stanford University start-up funds.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ferreira, J.P., Peacock, R.W.S., Lawhorn, I.E.B. et al. Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters. Syst Synth Biol 5, 131–138 (2011). https://doi.org/10.1007/s11693-011-9089-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11693-011-9089-0