Schaniel Christoph, Lee Dung-Fang, Lemischka Ihor R
Department of Gene and Cell Medicine, Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, USA.
Methods Enzymol. 2010;477:351-65. doi: 10.1016/S0076-6879(10)77018-X.
Embryonic stem cells (ESCs) have the ability to expand indefinitely in vitro and give rise to cells of all three germ layers as well as germ cells. For these reasons, ESCs hold great promise for biomedicine. In order to harness the potential of pluripotent cells, it is necessary to first understand the molecular mechanisms that control the pluripotent state. The discovery of RNA interference has made such functional analysis, even at high(er) throughput, possible. Here, we describe the methods used for high-throughput siRNA screening by high-content microscopy to identify gene products that regulate mouse ESC fate decision. In addition, we will describe the application of lentivirus-based shRNA knockdown to explore or validate the role of candidate genes in ESC pluripotency.
胚胎干细胞（ESCs）能够在体外无限扩增，并分化形成所有三个胚层的细胞以及生殖细胞。基于这些原因，胚胎干细胞在生物医学领域具有巨大的应用前景。为了充分利用多能干细胞的潜能，首先需要了解控制多能状态的分子机制。RNA干扰的发现使得即使在高通量情况下进行这种功能分析成为可能。在此，我们描述了通过高内涵显微镜进行高通量siRNA筛选以鉴定调控小鼠胚胎干细胞命运决定的基因产物所使用的方法。此外，我们还将描述基于慢病毒的shRNA敲低技术在探索或验证候选基因在胚胎干细胞多能性中的作用方面的应用。
