Abstract
Constitutive promoters are used routinely to drive ectopic gene expression. Here, we carried out a systematic comparison of eight commonly used constitutive promoters (SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems, and COPIA and ACT5C for Drosophila systems). We also included in the comparison the TRE promoter, which can be activated by the rtTA transcriptional activator in a doxycycline-inducible manner. To make our findings representative, we conducted the comparison in a variety of cell types derived from several species. We found that these promoters vary considerably from one another in their strength. Most promoters have fairly consistent strengths across different cell types, but the CMV promoter can vary considerably from cell type to cell type. At maximal induction, the TRE promoter is comparable to a strong constitutive promoter. These results should facilitate more rational choices of promoters in ectopic gene expression studies.
Highlights
Many constitutive promoters are used to drive ectopic gene expression in various in vitro and in vivo contexts
To facilitate more rational choices of promoters in ectopic gene expression studies, we decided to examine six constitutive promoters commonly used in mammalian systems, including the simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor 1a promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken b-Actin promoter coupled with CMV early enhancer (CAGG)
We quantitated GFP intensity by flow cytometry (Figure 1). It showed that UBC is consistently the weakest promoter in all the cell types while PGK is consistently weak, though typically stronger than UBC
Summary
Many constitutive promoters are used to drive ectopic gene expression in various in vitro and in vivo contexts. To facilitate more rational choices of promoters in ectopic gene expression studies, we decided to examine six constitutive promoters commonly used in mammalian systems, including the simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor 1a promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken b-Actin promoter coupled with CMV early enhancer (CAGG). For the six mammalian constitutive promoters, each one was inserted into a lentiviral expression vector in front of the GFP reporter [11].
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