Zheng Xiaofeng, Hu Guang
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, USA.
J Vis Exp. 2012 May 30(63):3987. doi: 10.3791/3987.
Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in (1,2)). To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells (3). The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region (4,5). Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation (6,7). As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity (5), and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level (3,8). Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal (3,8). Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.
多能性和自我更新是胚胎干细胞（ES细胞）的两个决定性特征。了解其潜在的分子机制将极大地促进ES细胞在发育生物学研究、疾病建模、药物发现和再生医学中的应用（参考文献(1,2)中的综述）。为了加速鉴定和表征ES细胞维持和自我更新的新型调节因子，我们开发了一种基于荧光报告基因的检测方法，使用Oct4GiP细胞定量测量小鼠ES细胞的自我更新状态(3)。Oct4GiP细胞在Oct4基因启动子区域的控制下表达绿色荧光蛋白（GFP）(4,5)。Oct4是ES细胞自我更新所必需的，在ES细胞中高度表达，并在分化过程中迅速下调(6,7)。因此，报告细胞中的GFP表达和荧光与ES细胞身份密切相关(5)，荧光激活细胞分选（FACS）分析可用于在单细胞水平上密切监测细胞的自我更新状态(3,8)。结合RNA干扰，Oct4GiP报告基因检测可用于快速鉴定和研究ES细胞维持和自我更新的调节因子(3,8)。与其他检测自我更新的方法相比，它更方便、灵敏、定量且成本更低。它可以在96孔或384孔板中进行，用于大规模研究，如高通量筛选或基因上位性分析。最后，通过使用其他谱系特异性报告ES细胞系，我们在此描述的检测方法也可以进行修改，以研究ES细胞分化过程中的命运决定。
