Pollex Tim, Piolot Tristan, Heard Edith
Mammalian Developmental Epigenetics Group, Institut Curie, CNRS UMR3215, INSERM U934, Paris, France.
Methods Mol Biol. 2013;1042:13-31. doi: 10.1007/978-1-62703-526-2_2.
Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.
胚胎干细胞的分化伴随着基因表达、染色质和染色体动力学的变化。这些变化最显著的例子之一是小鼠雌性胚胎干细胞分化时两条X染色体中的一条发生失活。除了少数例外，这些事件主要是在固定细胞中进行研究的。为了更好地理解分化过程中事件的动态、动力学和顺序，需要采用活细胞成像技术。在这里，我们描述了一种将活细胞成像与可用于固定细胞的技术（如RNA荧光原位杂交）相结合的方法，以便将基因座动态或亚核定位与基因表达等相关联。为了研究雌性胚胎干细胞中的基因座动态，我们构建了在X染色体失活中心含有TetO阵列的细胞系，X染色体失活中心是X染色体上调节X染色体失活的基因座，在与荧光蛋白融合的TetR表达时可被可视化。我们将使用这个系统详细阐述如何生成用于基因座动态活细胞成像的胚胎干细胞系，如何在活细胞成像前培养胚胎干细胞，并描述使用不同显微镜对胚胎干细胞进行典型活细胞成像的条件。此外，我们将解释在活细胞成像后如何应用RNA、DNA荧光原位杂交或免疫荧光来将基因表达与基因座动态相关联。
