Shiura Hirosuke, Sakata Yuka, Abe Kuniya, Sado Takashi
Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan.
Technology and Development Team for Mammalian Genome Dynamics, RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan.
Methods Mol Biol. 2018;1861:161-176. doi: 10.1007/978-1-4939-8766-5_13.
There are two modes of X chromosome inactivation (XCI) in the mouse. One mode is imprinted XCI: it is initiated at around the four-cell stage in favor of the paternal X chromosome, and is maintained in the extraembryonic tissues. The other mode is random XCI, which takes place in the epiblast lineage at the periimplantation stage. X-linked noncoding Xist RNA, which becomes upregulated on the X chromosome to be inactivated at the onset of XCI and plays a critical role in both imprinted and random XCI, and its accumulation in the nucleus have been referred to as one of the hallmarks of the presence of the inactivated X chromosome. RNA-FISH has therefore been an invaluable method for the study of XCI. As XCI status changes dynamically during periimplantation development in the mouse, analysis using samples from these developmental stages is absolutely necessary for elucidation of the molecular basis of XCI mechanisms. However, dissection of the embryos at around the periimplantation stages is not easy, and this impedes in vivo analysis of the kinetics of XCI. Here, we describe our methods for dissecting the periimplantation stage embryo and subsequent procedures for RNA-FISH and immunostaining.
在小鼠中存在两种X染色体失活（XCI）模式。一种模式是印记XCI：它在大约四细胞阶段开始，有利于父本X染色体，并在胚外组织中维持。另一种模式是随机XCI，它发生在着床前期的上胚层谱系中。X连锁非编码Xist RNA在XCI开始时在将被失活的X染色体上上调，并在印记和随机XCI中都起关键作用，其在细胞核中的积累被认为是失活X染色体存在的标志之一。因此，RNA荧光原位杂交（RNA-FISH）一直是研究XCI的一种非常有价值的方法。由于在小鼠着床前期发育过程中XCI状态动态变化，使用这些发育阶段的样本进行分析对于阐明XCI机制的分子基础绝对必要。然而，在着床前期左右解剖胚胎并不容易，这阻碍了对XCI动力学的体内分析。在这里，我们描述了我们解剖着床前期胚胎的方法以及随后进行RNA-FISH和免疫染色的步骤。
