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Differential transcriptional regulation of the nanog gene in chicken primordial germ cells and embryonic stem cells

Journal of Animal Science and Biotechnology 2021年第3期12卷 877-890页

作者： Hee Jung Choi So Dam Jin Deivendran Rengaraj Jin Hwa Kim Bertrand Pain Jae Yong Han Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences Seoul National UniversitySeoul 08826South Korea Univ Lyon UniversitéLyon 1INSERMINRAEStem Cell and Brain Research InstituteU1208USC136169500 BronFrance Institute for Biomedical Sciences Shinshu UniversityMinamiminowaNagano 399-4598Japan

Background:nanog is a core transcription factor(TF)in embryonic stem cells(ESCs)and primordial germ cells(PGCs).Regulation of the nanog gene by TFs,epigenetic factors,and autoregulatory factors is well characterized in ESCs,and transcriptional regulation of nanog is well established in these *** nanog plays a key role in germ cells,the molecular mechanism underlying its transcriptional regulation in PGCs has not been ***,we investigated the mechanism that regulates transcription of the chicken nanog(cnanog)gene in PGCs and ***:We first identified the transcription start site of cnanog by 5′-rapid amplification of cDNA ends PCR ***,we measured the promoter activity of various 5′flanking regions of cnanog in chicken PGCs and ESCs using the luciferase reporter *** expression required transcriptional regulatory elements,which were positively regulated by POU5F3(OCT4)and SOX2 and negatively regulated by TP53 in *** proximal region of the cnanog promoter contains a positive transcriptional regulatory element(CCAAT/enhancer-binding protein(CEBP)-binding site)in ***,small interfering RNA-mediated knockdown demonstrated that POU5F3,SOX2,and CEBP played a role in cell type-specific transcription of ***:We show for the first time that different trans-regulatory elements control transcription of cnanog in a cell type-specific *** finding might help to elucidate the mechanism that regulates cnanog expression in PGCs and ESCs.

关键词： Chicken Embryonic stem cells nanog gene Primordial germ cells Regulatory elements

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Construction of Prokaryotic Expression Vector of Mouse nanog gene and Its Expression

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Agricultural Sciences in China 2007年第4期6卷 487-492页

作者： LI Jun Lü Chang-rong DOU Lin DOU Zhong-ying Shaanxi Center of National Stem Cell Engineering ＆ Technology Center Northwest A＆F University Yangling 712100 P.R.China

The aim of this study is to construct a prokaryotic expression vector of mouse nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- nanog, was constructed successfully. The pGEX-KG-nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of nanog protein and for the preparation of polyclonal antibody.

关键词： nanog gene prokaryotic expression glutathione-S-transferase (GST) fusion protein mouse

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