title: Silencing of the expression of pluripotent driven-reporter genes stably transfected into human pluripotent cells.
abstract: OBJECTIVE
  Marking of human embryonic stem (ES) and embryonal carcinoma (EC) cells with pluripotent promoter-driven reporter gene cassettes provides an important tool for studies related to maintenance of pluripotency, cell differentiation and cell selection. OCT4, TERF1 and telomerase reverse transcriptase component (TERT) are considered as pluripotent marker genes since they are expressed in both human ES and EC cells and significantly downregulated during the differentiation process. Our aim was to use core promoter regions from such pluripotent genes to drive expression of reporter genes that would be suitable for human ES cell selection amongst differentiated cells.
  
  
  RESULTS
  Human ES and EC cells were stably transfected with a number of TERT, OCT4 and TERF1 promoter-driven EGFP or NTR gene cassettes. Gradual loss of reporter gene expression was observed from 24 h post-transfection during transient transfection studies, while almost complete loss of reporter expression was observed upon stable transfections. The loss of reporter gene expression was partly reversed by addition of a histone deacetylase inhibitor and a demethylating agent, suggesting that in vitro methylation of these exogenous constructs and the epigenetic architecture around the site of integration are likely to play a major role in their transcriptional activity. Inclusion of gene-regulatory elements in addition to the core promoters has been shown to minimize such effects and should be considered as an important strategy in such studies.
  
  
  CONCLUSIONS
  Together our data suggest that human ES and EC cells are able to silence pluripotent promoter-driven reporter genes with high efficiency. Whether differentiated cells derived from human ES and EC cells retain this activity is unknown and need to be investigated before large-scale comparative reporter-based transfection studies can be used as a tool in human embryonic stem cell biology.
authors.affiliation: North East Institute for Stem Cell Research, Newcastle University International Centre for Life, Newcastle NE13BZ, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Stewart
authors.forename: Chunbo
authors.initials: C
authors.lastname: Yang
authors.forename: George
authors.initials: G
authors.lastname: Anyfantis
authors.forename: Stefan
authors.initials: S
authors.lastname: Przyborski
authors.forename: Nicholas
authors.initials: N
authors.lastname: Hole
authors.forename: Tom
authors.initials: T
authors.lastname: Strachan
authors.forename: Miodrag
authors.initials: M
authors.lastname: Stojkovic
authors.forename: W Nicol
authors.initials: WN
authors.lastname: Keith
authors.forename: Lyle
authors.initials: L
authors.lastname: Armstrong
authors.forename: Majlinda
authors.initials: M
authors.lastname: Lako
mesh_terms: D019175:DNA Methylation; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection / Q000379:methods*