Department of Molecular Biology, Massachusetts General Hospital, Howard Hughes Medical Institute, Boston, Massachusetts, USA.
Nat Protoc. 2011 Mar;6(3):270-84. doi: 10.1038/nprot.2010.195. Epub 2011 Feb 10.
Dynamic reprogramming of the genome takes place during the gamete-to-embryo transition. This transition defines a period of continuous and global change but has been difficult to study because of extremely limited material and varying degrees of chromatin compaction. Improved methods of detecting chromatin and gene expression changes in the germ line and in the preimplantation embryo would greatly enhance the understanding of this crucial developmental transition. Here we describe a protocol developed and used by us that improves the sensitivity of existing fluorescence in situ hybridization (FISH) methods; the protocol described here has enabled us to visualize single-copy DNA targets and corresponding nascent RNA transcripts in preimplantation embryos and during spermatogenesis. Major improvements over alternative methods involve fixation and permeabilization steps. Chromatin epitopes can be visualized simultaneously by combining FISH with immunofluorescence; multicopy and repetitive element expression can also be reliably measured. This procedure (sample preparation and staining) requires 1-1.5 d to complete and will facilitate detailed examination of spatial relationships between chromatin epitopes, DNA and RNA during the dynamic transition from gamete to embryo.
基因组的动态重编程发生在配子到胚胎的转变过程中。这一转变定义了一个持续的、全球性的变化时期，但由于材料极其有限，以及染色质的不同程度的紧缩，使得研究变得困难。改进检测生殖细胞系和着床前胚胎中染色质和基因表达变化的方法将极大地增强对这一关键发育转变的理解。在这里，我们描述了一个由我们开发并使用的方案，该方案提高了现有荧光原位杂交（FISH）方法的灵敏度；这里描述的方案使我们能够在着床前胚胎和精子发生过程中可视化单拷贝 DNA 靶标和相应的新生 RNA 转录本。与替代方法相比，主要的改进涉及固定和透化步骤。通过将 FISH 与免疫荧光相结合，可以同时可视化染色质表位；也可以可靠地测量多拷贝和重复元件的表达。这个过程（样品制备和染色）需要 1-1.5 天才能完成，将有助于在配子到胚胎的动态转变过程中详细检查染色质表位、DNA 和 RNA 之间的空间关系。
