Zheng Xiaofeng, Hu Guang
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709, USA.
Methods Mol Biol. 2014;1150:163-73. doi: 10.1007/978-1-4939-0512-6_10.
Embryonic stem cells (ESCs) are characterized by two defining features: pluripotency and self-renewal. They hold tremendous promise for both basic research and regenerative medicine. To fully realize their potentials, it is important to understand the molecular mechanisms regulating ESC pluripotency and self-renewal. The development of RNA interference (RNAi) technology has revolutionized functional genetic studies in mammalian cells. In recent years, genome-wide RNAi screens have been adopted to systematically study ESC biology, and have uncovered many previously unknown regulators, including transcription factors, chromatin remodelers, and posttranscriptional modulators. Here, we describe a method for the identification of regulators of ESC pluripotency and self-renewal using RNAi screens, as well as assays for further validation and characterization of the identified candidates. With modifications, this method can also be adapted to study the fate specification events during ESC differentiation.
胚胎干细胞（ESCs）具有两个决定性特征：多能性和自我更新能力。它们在基础研究和再生医学领域都有着巨大的应用前景。为了充分发挥其潜力，了解调控胚胎干细胞多能性和自我更新的分子机制至关重要。RNA干扰（RNAi）技术的发展彻底改变了哺乳动物细胞中的功能基因研究。近年来，全基因组RNAi筛选已被用于系统地研究胚胎干细胞生物学，并发现了许多以前未知的调节因子，包括转录因子、染色质重塑因子和转录后调节因子。在这里，我们描述了一种使用RNAi筛选来鉴定胚胎干细胞多能性和自我更新调节因子的方法，以及用于进一步验证和表征已鉴定候选因子的检测方法。通过适当修改，该方法也可用于研究胚胎干细胞分化过程中的命运决定事件。
