Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression ...Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression of Nanog is very sensitive to ES cells differentiation, making Nanog one of the best markers to indicate the status of ES cells. In this study, we developed an efficient method to construct Nanog promoter driven EGFP reporter system based on the BAC homologous recombination. We further generated a Nanog-EGFP reporter mES cell line. This reporter mES cell line exhibited features similar to those of normal mES cells, and the EGFP reporter efficiently reflected the expression of Nanog, indi- cating the differentiation status of mES cells. We achieved a reliable experimental reporter system to research self-renewal and differentiation of mES cells. The system could facilitate research on culture system of mES cells and researches on the expression and regulation of Nanog and other related fac- tors in mES cells.展开更多
基金This research was supported by the Ministry of Science and Technology Grant （2001CB510106）；Science and Technology Plan of Beijing Municipal Government （H020220050290）；National Natural Science Foundation of China Awards for 0utstanding Young Scientists （30125022）；for Creative Research Groups （30421004）；Bill ＆ Melinda Gates Foundation Grant （37871） to H Deng.
文摘为人的胚胎的茎(ES ) 的自强和区别的能力细胞为对待类型 Idiabetes mellitus 为胰腺的贝它细胞的产生使他们成为潜在的来源。这里，我们报导一最新发展了并且有效方法，在aserum免费的系统执行了，区分进生产胰岛素的 cells.Activin A 的导致的人的 ES 房间它在起始的阶段被使用从人的 EScells 导致权威的内胚叶区别，是由权威的内胚叶标记 Sox17 和 Brachyury.Further 的表示检测了， all-trans retinoic 酸( RA )被用来支持胰腺的区别，由早胰腺的抄写因素 pdx1 和 hlxb9 的表示显示了。在成熟 inDMEM/F12 以后有 bFGF 和菸碱的没有浆液的媒介，区分的房间表示了小岛特定的标记象 C 肽，胰岛素，胰高血糖素和 glut2 那样。百分比 ofC-peptide-positive 房间超过了 15% 。由这些房间的胰岛素和 C 肽的分泌物在葡萄糖层次对应于变化。当移植了进肾的囊时， ofStreptozotocin (STZ ) 对待裸体老鼠，这些区分的人的 ES 房间熬过并且维持贝它房间标记基因的表示包括 C 肽， pdx1， glucokinase， nkx6.1， IAPP， pax6and Tcf1。百分之三十只移植裸体老鼠展出了 stableeuglycemia 的明显的恢复；并且改正的显型被支撑超过六个星期。我们的新方法为学习人的胰开发的机制提供一个有希望的试管内区别模特儿并且说明为类型 Idiabetes mellitus 的处理使用人的 ES 房间的潜力。
基金This research was supported by grants from the Ministry of Ed- ucation （705001）, National Basic Research Program of China （973 Program 2009CB941200）, National Natural Science Foundation of China （30830061 and 30421004）, and a 111 project to H Deng. We thank Dr Tak Wah Mak （University of Alberta, Canada） for kindly providing the Ptern mice, Dr Guoqiang Gu （Vanderbilt University, USA） for kindly providing the plasmid of Pdxl-Cre, and Dr C Wright （Vanderbilt University, USA） for the PDX1 antibody. We thank the Model Animal Research Center of Nanjing University for B6 129-Gt（ROSA）26Sor tm/Sho/J mice and the Research Center for Proteome Analysis for proteomics analysis. We thank Dr Matt Stremlau, Dr Hui Zhang, Jun Cai, Han Qin, Jian Li, Yan Shi, Haisheng Zhou, and Fei Ye for their critical reading of the manu- script. We also thank Wei Jiang, Yushan Guo, Jie Yang, Chengyan Wang, Hui Zhang, and other colleagues in our laboratory for providing technical assistance and advice during the experiments.
基金Supported by the National Natural Science Foundation of China (Grant No. 30421004)
文摘Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression of Nanog is very sensitive to ES cells differentiation, making Nanog one of the best markers to indicate the status of ES cells. In this study, we developed an efficient method to construct Nanog promoter driven EGFP reporter system based on the BAC homologous recombination. We further generated a Nanog-EGFP reporter mES cell line. This reporter mES cell line exhibited features similar to those of normal mES cells, and the EGFP reporter efficiently reflected the expression of Nanog, indi- cating the differentiation status of mES cells. We achieved a reliable experimental reporter system to research self-renewal and differentiation of mES cells. The system could facilitate research on culture system of mES cells and researches on the expression and regulation of Nanog and other related fac- tors in mES cells.
