Ranisavljevic Noémie, Okamoto Ikuhiro, Heard Edith, Ancelin Katia
Unité de Génétique et Biologie du Développement, Institut Curie, PSL Research University, CNRS UMR 3215, INSERM U934, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto university, yoshida-konoe-cho, saikyo-ku, Kyoto 606-0581, Japan.
Methods Mol Biol. 2017;1605:133-145. doi: 10.1007/978-1-4939-6988-3_9.
Characterizing the maternal-to-zygotic transition (MZT) is a central question in embryogenesis, and is critical for our understanding of early post-fertilization events in mammals. High-throughput RNA sequencing (RNA Seq) of mouse oocytes and early embryos has recently revealed that elaborate transcription patterns of genes and repeats are established post-fertilization. This occurs in the context of the gradually depleted maternal pool of RNA provided by the oocyte, which can confound the accurate analysis of the zygotic genome activation when the mRNA population is sequenced. In this context, and given the limited amounts of material available from embryos, particularly when studying mutants, as well as the cost of sequencing, an alternative, complementary single cell approach is RNA FISH. This approach can assay the expression of specific genes or genetic elements during preimplantation development, in particular during the MZT. Here, we describe how RNA FISH can be applied to visualize nascent transcription at specific genomic loci in embryos at different stages of preimplantation development and also discuss possible analytical methods of RNA FISH data.
表征母源-合子转变（MZT）是胚胎发育中的核心问题，对于我们理解哺乳动物受精后的早期事件至关重要。最近，对小鼠卵母细胞和早期胚胎进行的高通量RNA测序（RNA Seq）显示，受精后会建立基因和重复序列的精细转录模式。这一过程发生在卵母细胞提供的母源RNA池逐渐耗尽的背景下，当对mRNA群体进行测序时，这可能会混淆对合子基因组激活的准确分析。在这种情况下，考虑到从胚胎中获取的材料数量有限，特别是在研究突变体时，以及测序成本，一种替代的、互补的单细胞方法是RNA荧光原位杂交（FISH）。这种方法可以检测着床前发育过程中，特别是MZT期间特定基因或遗传元件的表达。在这里，我们描述了如何应用RNA FISH来可视化着床前发育不同阶段胚胎中特定基因组位点的新生转录，并讨论了RNA FISH数据的可能分析方法。
