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...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.展开更多
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 i...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 cells.Although NANOG plays a key role in germ cells,the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied.Therefore,we investigated the mechanism that regulates transcription of the chicken NANOG(cNANOG)gene in PGCs and ESCs.Results:We first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis.Then,we measured the promoter activity of various 5′flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay.cNANOG expression required transcriptional regulatory elements,which were positively regulated by POU5F3(OCT4)and SOX2 and negatively regulated by TP53 in PGCs.The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element(CCAAT/enhancer-binding protein(CEBP)-binding site)in ESCs.Furthermore,small interfering RNA-mediated knockdown demonstrated that POU5F3,SOX2,and CEBP played a role in cell type-specific transcription of cNANOG.Conclusions:We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner.This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.展开更多
Background: The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells(SSCs) via interacting with transcription factor...Background: The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells(SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp(Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements.Results: Rohu Nanog(LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetical y, it was closely related to freshwater counterparts. Protein sequence(386 AA of42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis-à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements.Conclusion: The molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.展开更多
文摘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.
基金This work was supported by a National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIP)[2015R1A3A2033826]and[2018R1D1A1B07049376].
文摘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 cells.Although NANOG plays a key role in germ cells,the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied.Therefore,we investigated the mechanism that regulates transcription of the chicken NANOG(cNANOG)gene in PGCs and ESCs.Results:We first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis.Then,we measured the promoter activity of various 5′flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay.cNANOG expression required transcriptional regulatory elements,which were positively regulated by POU5F3(OCT4)and SOX2 and negatively regulated by TP53 in PGCs.The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element(CCAAT/enhancer-binding protein(CEBP)-binding site)in ESCs.Furthermore,small interfering RNA-mediated knockdown demonstrated that POU5F3,SOX2,and CEBP played a role in cell type-specific transcription of cNANOG.Conclusions:We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner.This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.
基金provided by grant from Indian Council of Agricultural Research (ICAR) and Department of Biotechnology(DBT),Government of India
文摘Background: The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells(SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp(Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements.Results: Rohu Nanog(LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetical y, it was closely related to freshwater counterparts. Protein sequence(386 AA of42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis-à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements.Conclusion: The molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.
