Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.
Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.
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DOI：
10.1038/nprot.2010.195
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发表时间：
2011-03
期刊：
影响因子：
14.8
通讯作者：
Lee, Jeannie T.
中科院分区：
文献类型：
--
作者：
Namekawa, Satoshi H.;Lee, Jeannie T.
基因组的动态重新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和全球变化的时期，但由于材料极其有限和染色质压缩程度不一，一直难以研究。改进检测胚系和植入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，我们描述了一种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可视化植入前胚胎和精子发生期间的单拷贝DNA靶标和相应的新生RNA转录本。与其他方法相比，主要的改进包括固定和渗透步骤。通过结合FISH和免疫荧光，可以同时显示染色质表位；还可以可靠地测量多拷贝和重复元件的表达。这一过程(样品制备和染色)需要1-1.5d的时间来完成，并将有助于详细检查从配子到胚胎的动态转变过程中染色质表位、DNA和RNA之间的空间关系。
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.
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影响因子：
64.8
作者：
COOPER, DW
通讯作者：
COOPER, DW
影响因子：
16.8
作者：
Brykczynska, Urszula;Hisano, Mizue;Peters, Antoine H. F. M.
通讯作者：
Peters, Antoine H. F. M.
DOI：
10.1073/pnas.0406325101
发表时间：
2004-11-23
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
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作者：
Silva, J;Mak, W;Brockdorff, N
通讯作者：
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影响因子：
12.4
作者：
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通讯作者：
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