The synergistic effect of 5Azadc and TSA on maintenance of pluripotency of chicken ESCs by overexpression of NANOG gene

In Vitro Cell Dev Biol Anim. 2016 Apr;52(4):488-96. doi: 10.1007/s11626-015-9993-9. Epub 2016 Jan 28.


NANOG is a transcription factor that functions in embryonic stem cells (ESCs) and a key factor in maintaining pluripotency. Here, we cloned the NANOG gene promoter from the Rugao yellow chicken and constructed a dual luciferase reporter vector to detect its transcriptional activity and analyze the effects of 5-aza-2'-deoxycytidine (5-Azadc) and trichostatin A (TSA) on NANOG promoter activity and ESC pluripotency maintenance in vitro. NANOG transcriptional activity was enhanced when 5-Azadc and TSA were used alone or together, suggesting the possibility of elevated methylation of the CpG island in the NANOG regulatory region. When ESCs were cultured in basic medium with 5-Azadc and TSA in vitro, significantly more cell colonies were maintained in the 5-Azadc + TSA group than in the control group, which had many differentiated cells and few cell colonies after 6 d of induction. On the tenth day of induction, the cells in the control group fully differentiated and no cell colonies remained, but many cell colonies were present in the 5-Azadc + TSA group. The expression of NANOG in the cell colonies was confirmed by indirect immunofluorescence. Furthermore, ESCs could be passaged to the 12th generation under 5-Azadc and TSA treatment and maintained their pluripotency. Thus, we showed that 5-Azadc and TSA can effectively maintain chicken ESC pluripotency in vitro by increasing NANOG gene expression.

Keywords: Dual luciferase reporter gene assay; ESC pluripotency; NANOG gene; Promoter.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Azacitidine / analogs & derivatives*
  • Azacitidine / pharmacology
  • Cell Differentiation / drug effects
  • Cell Line
  • Chickens
  • Cloning, Molecular
  • Computational Biology
  • DNA Methylation / drug effects
  • DNA Methylation / genetics
  • Decitabine
  • Drug Synergism
  • Embryonic Stem Cells / drug effects
  • Embryonic Stem Cells / metabolism
  • Fluorescent Antibody Technique
  • Gene Expression Regulation / drug effects*
  • Hydroxamic Acids / pharmacology*
  • Nanog Homeobox Protein / genetics*
  • Nanog Homeobox Protein / metabolism
  • Pluripotent Stem Cells / drug effects
  • Pluripotent Stem Cells / metabolism*
  • Promoter Regions, Genetic


  • Hydroxamic Acids
  • Nanog Homeobox Protein
  • trichostatin A
  • Decitabine
  • Azacitidine