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Quantitative Comparison of Constitutive Promoters in Human ES cells

Författare:
  • Karin Norrman
  • Yvonne Fischer
  • Blandine Bonnamy
  • Fredrik Wolfhagen Sand
  • Philippe Ravassard
  • Henrik Semb
Publiceringsår: 2010
Språk: Engelska
Publikation/Tidskrift/Serie: PLoS One
Volym: 5
Nummer: 8
Dokumenttyp: Artikel
Förlag: PLoS

Sammanfattning

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

Disputation

Nyckelord

  • Medicine and Health Sciences

Övrigt

Published
Yes
  • ISSN: 1932-6203

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