Quantitative Comparison of Constitutive Promoters in Human ES cells

Abstract

BackgroundConstitutive 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 FindingsWe 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/SignificanceThe 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.

A Stem Cell-Seeded Nanofibrous Scaffold for Auditory Nerve Replacement

“…We first established a plasmid vector with an optimal promoter for expression in human stem cells. In a previous study using lentivirus reporters expressed in hESCs, EF1α was identified as producing the most robust and stable gene expression during differentiation programs (Norrman et al, 2010). We found this to be true as well, when comparing GFP reporter expression in H7 cells transiently transfected with plasmids driven by CAG (chick beta-actin enhancer), CMV, and EF1α.…”

Safeguarding clinical translation of pluripotent stem cells with suicide genes

“…They usually achieve high levels of gene expression in most cell types. 26 However, these promoters may be transcriptional silenced due to extensive methylation. Previous studies have also shown that elongation factor 1α (EF1α) and constitutive cytomegalovirus (CMV) enhancer/chicken β-actin promoter (CAGG promoters are consistently strong in all mammalian cell types tested, whereas the CMV promoter is the most variable, being very strong in some cell types and rather weak in others.…”

“…27 Norrman et al found that ACTB (human β-actin promoter), EF1α and PGK promoters showed stable activities during longterm culture of undifferentiated hESCs. 26 Therefore, a promoter that can mediate stable, highly active suicide gene expression in human PSCs should be chosen for the clinical translation of hiPSCs in order to control wayward pluripotent stem cells and their progenies in vivo.…”

Expression of Non-Native Genes in a Surrogate Host Organism

“…For this reason, the selection of an appropriate promoter should be determined on a case-by-case basis. Recent studies have systematically compared many of the commonly used promoters in a variety of cell types (Norrman et al, 2010;Qin et al, 2010) (Figure 4). These types of references are an excellent source of information when designing constructs with specific expression needs.…”