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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
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
PLoS ONE
volume
5
issue
8
article number
e12413
publisher
Public Library of Science (PLoS)
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
2016-04-04 09:10:39
date last changed
2022-08-23 05:57:40
@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.}},
  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 (PLoS)}},
  series       = {{PLoS ONE}},
  title        = {{Quantitative comparison of constitutive promoters in human ES cells.}},
  url          = {{https://lup.lub.lu.se/search/files/5252759/1692105.pdf}},
  doi          = {{10.1371/journal.pone.0012413}},
  volume       = {{5}},
  year         = {{2010}},
}