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Quantitative comparison of constitutive promoters in human ES cells.

Norrman, Karin LU ; Fischer, Yvonne LU ; Bonnamy, Blandine; Wolfhagen Sand, Fredrik LU ; Ravassard, Philippe and Semb, Henrik LU (2010) In PLoS One 5(8).
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 β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene... (More)
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. (Less)
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author
organization
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Contribution to journal
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published
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in
PLoS One
volume
5
issue
8
publisher
Public Library of Science
external identifiers
  • WOS:000281292100013
  • PMID:20865032
  • Scopus:77957888099
ISSN
1932-6203
DOI
10.1371/journal.pone.0012413
language
English
LU publication?
yes
id
e32a5973-6f7a-48c4-93f9-1659e7a98ee8 (old id 1687907)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/20865032?dopt=Abstract
date added to LUP
2010-10-06 08:19:59
date last changed
2017-01-01 07:43:59
@article{e32a5973-6f7a-48c4-93f9-1659e7a98ee8,
  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 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.},
  articleno    = {e12413},
  author       = {Norrman, Karin and Fischer, Yvonne and Bonnamy, Blandine and Wolfhagen Sand, Fredrik and Ravassard, Philippe and Semb, Henrik},
  issn         = {1932-6203},
  language     = {eng},
  number       = {8},
  publisher    = {Public Library of Science},
  series       = {PLoS One},
  title        = {Quantitative comparison of constitutive promoters in human ES cells.},
  url          = {http://dx.doi.org/10.1371/journal.pone.0012413},
  volume       = {5},
  year         = {2010},
}