title: The synergistic effect of 5Azadc and TSA on maintenance of pluripotency of chicken ESCs by overexpression of NANOG gene.
abstract: 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.
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zuo
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Lian
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Beibei
authors.initials: B
authors.lastname: Tang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Tianrong
authors.initials: T
authors.lastname: Xiao
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
authors.forename: Man
authors.initials: M
authors.lastname: Wang
authors.affiliation: Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou, 225125, Jiangsu Province, People's Republic of China.
authors.forename: Kehua
authors.initials: K
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China. 744366503@qq.com.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China. 744366503@qq.com.
authors.forename: Bichun
authors.initials: B
authors.lastname: Li
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China. ynzhang@yzu.edu.cn.|Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China. ynzhang@yzu.edu.cn.
authors.forename: Yani
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D019295:Computational Biology; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D000077209:Decitabine; D004357:Drug Synergism; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects*; D006877:Hydroxamic Acids / Q000494:pharmacology*; D000071317:Nanog Homeobox Protein / Q000235:genetics* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic
chemical_list: D006877:Hydroxamic Acids; D000071317:Nanog Homeobox Protein; C012589:trichostatin A; D000077209:Decitabine; D001374:Azacitidine
keywords: Dual luciferase reporter gene assay; ESC pluripotency; NANOG gene; Promoter
references.citation: Gene. 1999 Nov 15;240(1):1-12
references.pmid: 10564807
references.citation: Cell Res. 2005 May;15(5):317-24
references.pmid: 15916719
references.citation: Biochem Biophys Res Commun. 2011 Aug 19;412(1):175-81
references.pmid: 21810410
references.citation: Bioinformatics. 2002 Nov;18(11):1427-31
references.pmid: 12424112
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10294-9
references.pmid: 16801560
references.citation: Development. 2006 Mar;133(6):1193-201
references.pmid: 16501172
references.citation: Biochem J. 2003 Mar 15;370(Pt 3):737-49
references.pmid: 12429021
references.citation: Cell Res. 2007 Jan;17 (1):42-9
references.pmid: 17211451
references.citation: Cell. 2009 Aug 21;138(4):722-37
references.pmid: 19703398
references.citation: Stem Cells Dev. 2010 Jul;19(7):979-88
references.pmid: 19845468
references.citation: Biochem Pharmacol. 2007 May 1;73(9):1297-307
references.pmid: 17276411
references.citation: Cell. 2003 May 30;113(5):643-55
references.pmid: 12787505
references.citation: Development. 2014 Jul;141(14 ):2770-9
references.pmid: 25005472
references.citation: Curr Biol. 2011 Jan 11;21(1):65-71
references.pmid: 21194951
references.citation: Oncogene. 2016 Mar 10;35(10 ):1334-40
references.pmid: 26073077
references.citation: Stem Cell Res. 2015 Mar;14(2):224-37
references.pmid: 25702531
references.citation: J Biol Chem. 2012 Apr 6;287(15):12417-24
references.pmid: 22315219
references.citation: Science. 2007 Dec 21;318(5858):1917-20
references.pmid: 18029452
references.citation: Dev Biol. 1976 Apr;49(2):321-37
references.pmid: 944662
references.citation: Dev Biol. 2014 Aug 15;392(2):182-92
references.pmid: 24929238
references.citation: Philos Trans R Soc Lond B Biol Sci. 2011 Aug 12;366(1575):2222-9
references.pmid: 21727127
references.citation: Cell. 2003 May 30;113(5):631-42
references.pmid: 12787504
references.citation: Stem Cells. 2010 Nov;28(11):1961-9
references.pmid: 20872845
references.citation: Stem Cells. 2008 Nov;26(11):2791-9
references.pmid: 18757296
references.citation: Biochem Biophys Res Commun. 2005 Dec 16;338(2):1098-102
references.pmid: 16259959
references.citation: Dev Dyn. 2004 May;230(1):187-98
references.pmid: 15108323
references.citation: Stem Cells. 2009 Sep;27(9):2114-25
references.pmid: 19522013