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Background  Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking.    Methodology/Principal Findings  We have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies.    Conclusion/Significance  The ACTB, EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75–80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs.

Quantitative Comparison of Constitutive Promoters in Human ES cells