5-Azadc 和TSA对鸡Nanog基因启动活性及ESC多能性维持的影响

doi:10.11843/j.issn.0366-6964.2015.12.008

Abstract

Abstract:

The aim of this study was to clone Nanog gene promoter of chicken and construct the expression vector containing dual luciferase report gene to analyze the promoter activity，find out its core regulatory region，explore the effect of 5-Azadc(5-Aza-2′-deoxycytidine) and TSA(Trichostatin A)on its promoter activity and ESC pluripotent maintaining in vitro condition，so as to elucidate preliminarily the regulating mechanisms of Nanog gene and provide the theory basis for the future research.The different length fragments Nanog gene promoter was amplified by PCR technology and cloned into pGL3-basic vector or pEGFP-N1 directly to construct a recombinant vector.After the recombinant vector was transfected into DF-1 cells for 24 h and the protein was collected，the transcriptional activity of Nanog gene was measured by dual luciferase assay system to search for the basic transcriptional regulatory region；The recombinant vector was transfected into DF-1 cells，and methylation inhibitor 5-Azadc or histone deacetylase inhibitor TSA was added to detect its effect on the promoter activity.The third generation ESC was selected and grown into the medium supplemented with the optimum concentration of 5-Azadc and TSA，the cells were observed every 2 d to analyze the pluripotent maintaining of ESC in vitro culture condition，and indirect immune fluorescence of SSEA-1 was detected on the tenth days induction.The results showed that 3 dual luciferase report gene expression vector was constructed successfully.The strongest dual luciferase activity was shown by pGL3/1967；the transcription activity of Nanog gene was enhanced when 5-Azadc and TSA was added single or together.ESC was cultured in the medium with 5-Azadc and TSA in vitro，a great number of cells differentiated after 6 d of induction and cell colony was not maintained in control group，while the cell colony in 5-Azadc and TSA induction group was significant higher than the control group；On the tenth days induction，the cells in control group were fully differentiated，there were still a large number of cell clone in the 5-Azadc + TSA group，the number of colony were significantly more than the other 3 groups；the results of indirect immunofluorescence showed there was no cell colony in the control group，but more colonies were observed in the 5-Azadc and TSA induction group.It further showed that 5-Azadc and TSA could effectively maintain ESC pluripotence of chicken in vitro culture condition by increasing the expression of Nanog gene.

CLC Number:

S831.2

ZHANG Ya-ni，WANG Ying-jie，ZUO Qi-sheng，LI Dong，ZHANG Lei，TANG Bei-bei，LIAN Chao，BI Yu-lin，ZHANG Wen-hui，LI Bi-chun. Effects of 5-Azadc and TSA Induction on Promoter Activity of Nanog and Pluripotency Maintaining in Chicken[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2176-2184.

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Effects of 5-Azadc and TSA Induction on Promoter Activity of Nanog and Pluripotency Maintaining in Chicken

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