Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

• Published in: Systems and synthetic biology 2011-12, Vol.5 (3-4), p.131-138
• Main Authors: Ferreira, Joshua P., Peacock, Ryan W. S., Lawhorn, Ingrid E. B., Wang, Clifford L.
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Biomedicine; Cellular biology; Computational Biology/Bioinformatics; Elongation; Gene expression; Human cytomegalovirus; Metabolomics; Methodology; Methodology Article; Mutagenesis; Plasmids; Systems Biology
• ISSN: 1872-5325; 1872-5333
• DOI: 10.1007/s11693-011-9089-0

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.