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

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (12): 2176-2184.doi: 10.11843/j.issn.0366-6964.2015.12.008

5-Azadc 和TSA对鸡Nanog基因启动活性及ESC多能性维持的影响

张亚妮，王颖洁，左其生，李东，张蕾，汤贝贝，连超，毕瑜林，张文慧，李碧春^*

（扬州大学动物科学与技术学院江苏省动物遗传繁育与分子设计重点实验室，扬州 225009）

收稿日期:2015-02-11 出版日期:2015-12-23 发布日期:2015-12-23
通讯作者: 李碧春，教授，博士，主要从事动物胚胎工程与遗传工程研究，E-mail：yubcli@yzu.edu.cn
作者简介:张亚妮（1977-），女，陕西渭南人，副教授，博士，主要从事动物胚胎工程与遗传工程研究，E-mail：ynzhang@yzu.edu.cn
基金资助:
国家自然科学基金（31301959；31272429；31472087）；高等学校博士学科点专项科研基金（20123250120009）；中国博士后基金(2012M511326)；江苏高校优势学科建设工程资助项目

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

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^*

(College of Animal Science and Technology，Yangzhou University，Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province，Yangzhou 225009，China)

Received:2015-02-11 Online:2015-12-23 Published:2015-12-23

摘要/Abstract

摘要：

旨在克隆如皋黄鸡Nanog基因启动子，并构建含有双荧光素酶报告基因的表达载体进行启动子活性分析，找出该基因启动子的核心调控区；探索甲基化抑制剂5-Azadc (5-Aza-2′-deoxycytidine)或曲古抑菌素 (Trichostatin A，TSA)对Nanog基因启动活性及其ESC体外培养条件下多能性维持的影响，为初步阐明Nanog基因的表达调控机制提供理论依据。PCR扩增Nanog基因不同长度片段的启动子序列，定向克隆至pGL3-basic载体和pEGFP-N1构建重组载体；将重组载体转染DF-1细胞后，48 h收集蛋白，采用双荧光素酶报告基因检测系统测定其转录活性，确定基本转录调控区；将重组载体转染DF-1细胞后添加5-Azadc或TSA对Nanog基因进行诱导表达，并通过双荧光素酶报告基因检测系统，分析其对Nanog基因启动子活性的影响。选取生长至第3代的ESC，培养基中添加最适浓度的 5-Azadc和TSA，每隔两天观察细胞，分析其对ESC体外多能性维持的影响，并对诱导至第10天的细胞进行间接免疫荧光检测。结果表明，成功构建3个片段的双荧光素酶报告基因表达载体；双荧光素酶活性检测结果显示，pGL3/1967活性最强；分别添加或者联合添加5-Azadc和TSA均可使Nanog基因的转录活性增强；5-Azadc和TSA诱导对ESC体外培养条件下多能性维持的影响结果显示，诱导6 d 后，对照组细胞大量分化，细胞克隆无法维持，而5-Azadc和TSA诱导组克隆明显，5-Azadc+TSA联合诱导组细胞克隆数量显著多于对照组；诱导至第10天，对照组细胞完全分化，没有细胞克隆，而诱导组存在大量细胞克隆，联合诱导组克隆数量显著多于其他两组；SSEA-1间接免疫荧光结果显示，对照组没有明显的ESC克隆，而5-Azadc组和联合诱导组细胞克隆明显，综上表明，5-Azadc和TSA可有效地通过提高Nanog基因的表达而维持鸡ESC在体外培养过程中的多能性。

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.

中图分类号:

S831.2

张亚妮，王颖洁，左其生，李东，张蕾，汤贝贝，连超，毕瑜林，张文慧，李碧春. 5-Azadc 和TSA对鸡Nanog基因启动活性及ESC多能性维持的影响[J]. 畜牧兽医学报, 2015, 46(12): 2176-2184.

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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5-Azadc 和TSA对鸡Nanog基因启动活性及ESC多能性维持的影响

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

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