使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测
Summary
我们展示了染色质免疫沉淀（ChIP）的方法来识别组织特异性基因表达的组织特异性基因的小鼠胚胎组织中的发病因素相互作用期间或之后。此协议应广泛适用的组织特异性基因的激活的研究，因为它在正常胚胎发育过程中发生。
Abstract
染色质免疫沉淀（ChIP）是一个强大的工具，以确定蛋白质：发生在活细胞的情况下1-3的染色质相互作用。这项技术已被广泛利用组织培养细胞中，并在较小程度上，在基层组织。啮齿类动物的胚胎组织，特别是在发展的早期，芯片的应用，是由组织的数量有限，异质性的胚胎细胞和组织类型复杂。在这里，我们提出了一种方法来执行芯片采用一个分离的胚胎每天8.5（E8.5）胚胎。剪切从单一E8.5胚胎的染色质可分为五个等份，它允许为控制和特定蛋白质的调查研究者足够的材料：染色质相互作用。
我们已经利用这种技术来开始：在组织特异性基因表达程序的规范文件蛋白质染色质相互作用。类型的细胞在胚胎的异质性必然限制了这项技术的应用，因为其结果是不区分的相互作用是否发生在所有检测的蛋白质：染色质相互作用的一个子集，或一个单一的细胞类型（S）。然而，考试期间或之后的组织特异性基因的组织特异性的基因表达发病原因有两个可行的。首先，一定免疫组织特定的因素隔离开来表达的细胞类型，其中的因素是染色质。其次，含有基因激活相关的翻译后修改的共激活因子和组蛋白的免疫应该只被发现在基因的细胞类型的基因和基因调控序列，或已被激活。这项技术应该适用于大多数组织特异性基因的激活事件的研究。
在这个例子中所述，我们利用E8.5和E9.5小鼠胚胎的研究在骨骼肌特定基因的启动子具有约束力的因素。体节，躯干和四肢骨骼肌肉就会形成易制毒化学组织，目前在E8.5 – 9.5 4,5。肌细胞生成素是一种调节因子骨骼肌分化6-9所需。数据表明，肌细胞生成素是与它自己的启动子在E8.5和E9.5胚胎。由于肌细胞生成素只在体节在这个发展的6,10阶段表示，数据表明，肌细胞生成素的相互作用与它自己的启动已经发生E8.5胚胎骨骼肌中的前体细胞。
Protocol
1。胚胎隔离 注 ：所有涉及小鼠的操作，应当按照相应的动物护理和使用的政策和协议执行检查雌性小鼠交配塞存在交配后的上午，他们放置在不同的笼子分开根根男性的交配女性。交配塞观察的一天中午，被认为是发展的0.5天（E0.5）胚胎。 在E8.5，（或所需的阶段，如果有不同的），牺牲鼠标使用经批准的协议。 湿安乐死动物用70％乙醇?…
Discussion
在所描述的芯片协议，我们将展示生肌调节肌细胞生成素是在骨骼肌易制毒化学组织目前在单E8.5和E9.5胚胎肌细胞生成素推动者。此前的研究已经广泛的特点肌细胞生成素E盒含序列的结合，从最初在体外凝胶迁移实验利用体外翻译或细菌产生的肌细胞生成素和放射性标记的DNA编码的靶基因序列11-20监管的相关部分。传统的芯片研究已经证明了肌细胞生成素具有约束力的组织文…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院R01 GM56244安仁，其中包括通过美国复苏与再投资法“，2009年授予的资金支持，由美国国立卫生研究院R01 GM87130 JARP
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| ChIP Assay Kit | Upstate Cell Signaling Solutions, Millipore | 17-295 | ||
| Collagenase Type II | Invitrogen | 17101015 | Dilution by 1 x PBS | |
| Dulbecco’s modified eagle medium (DMEM) | Gibco Labs, Invitrogen | 12100-061 | High glucose content | |
| Dulbecco’s phosphate buffered saline 1X (DPBS) | Gibco Labs, Invitrogen | 14190-144 | Calcium chloride free, Magnesium chloride free | |
| Fetal bovine serum (FBS) | Mediatech, Inc. | 35-010-CV | ||
| Gel extraction kit | QIAquick | 28704 | 50 reaction kit | |
| Penicillin/streptomycin stock solution | Gibco Labs, Invitrogen | 5000 μg/ml concentration | ||
| Protease Inhibitor Cocktail | Sigma-Aldrich | P8340 | ||
| Salmon sperm DNA /Protein A agarose | Millipore | 16-157 | ||
| myogenin antibody | Santa Cruz Biotechnology, Inc. | sc-576 | ||
| Normal rabbit IgG | Millipore | 12-370 | ||
| Platinum PCR Supermix | Invitrogen | 11306-016 | ||
| GoTaq Q-PCR master mix | Promega | A6001 |
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Cite This Article
Cho, O. H., Rivera-Pérez, J. A., Imbalzano, A. N. Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos. J. Vis. Exp. (50), e2677, doi:10.3791/2677 (2011).