Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter

Parts used

Key findings

• Among six constitutive promoters compared side by side in a lentiviral GFP reporter across eight mammalian cell lines (129TF, MEF, C2C12, MSC, MRC5, HT1080, 293T, and CMMT), the human ubiquitin C promoter (UBC) is consistently the weakest driver of expression, and the mouse phosphoglycerate kinase 1 promoter (PGK) is also consistently weak, though typically stronger than UBC. The human elongation factor 1α promoter (EF1A) and the CAGG promoter (chicken β-actin promoter fused to the CMV immediate-early enhancer) are consistently the strongest of the six, with EF1A slightly stronger in some cell types and slightly weaker in others relative to CAGG, while the SV40 early promoter is fairly strong but generally somewhat weaker than EF1A or CAGG.
• The cytomegalovirus immediate-early promoter (CMV) is the most cell-type-variable of the six mammalian constitutive promoters tested, driving very strong GFP expression in 293T and CMMT cells but comparatively weak expression in MRC5 human fibroblasts and rat mesenchymal stem cells (MSC), consistent with previously reported silencing of this promoter in certain cell types.
• In Drosophila melanogaster cell lines (ML-DmBG3-c2 and S2R+), delivered by transient transfection rather than lentiviral transduction because lentiviral infection is lethal to these cells, the copia transposon promoter (COPIA) and the actin 5C promoter (ACT5C) drive similar levels of GFP expression, with ACT5C slightly stronger than COPIA.
• Five of the six mammalian constitutive promoters (CMV, UBC, EF1A, PGK, and CAGG) retain above-background activity when tested by transient transfection in Drosophila C2 and S2R+ cells, despite having evolved for mammalian transcriptional machinery; only the SV40 promoter fails to drive detectable expression in these fly cell lines. PGK, one of the weakest promoters in mammalian cells, becomes disproportionately active in Drosophila cells, reaching an expression level approaching that of the native Drosophila promoters COPIA and ACT5C.
• The tetracycline-responsive element promoter (TRE), driven by the reverse tetracycline-controlled transactivator (rtTA) expressed from an EF1A promoter, produces essentially undetectable GFP expression in the absence of doxycycline across all eight mammalian and two Drosophila cell types tested, but at maximal doxycycline induction its output rises to a level comparable to, or only slightly below, that of the strongest constitutive promoters, EF1A and CAGG, in the same cell type.
• Doxycycline sensitivity of the rtTA-TRE system varies by cell type: CMMT cells reach detectable GFP induction at lower doxycycline concentrations than C2C12 cells, and most cell types reach maximal TRE-driven expression at approximately 110 ng/ml doxycycline, though some cell types, such as rat mesenchymal stem cells (MSC), plateau at a lower concentration.

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.