Quantitative comparison of constitutive promoters in human ES cells.

ABSTRACT:

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 ?-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1?, (EF1?), 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, EF1? 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 EF1? 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.

SUBMITTER: Norrman K 

PROVIDER: S-EPMC2928720 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature