Chromatin immunoprecipitation for studying transcriptional regulation in Xenopus oocytes and tadpoles.
Chromatin immunoprecipitation for studying transcriptional regulation in Xenopus oocytes and tadpoles.
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DOI：
10.1007/978-1-59745-000-3_12
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发表时间：
2006
影响因子：
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通讯作者：
D. Stewart;A. Tomita;Yunbo Shi;J. Wong
中科院分区：
文献类型：
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作者：
D. Stewart;A. Tomita;Yunbo Shi;J. Wong
了解发育过程中基因表达的准确时间和空间调控需要了解所涉及的转录因子和辅因子的范围及其与染色质的功能相互作用。染色质免疫沉淀 (ChIP) 已成为一项强大的技术，使我们能够做到这一点。典型的 ChIP 检测包括 (1) 用甲醛处理细胞或组织，以将染色质相关蛋白快速交联到 DNA，(2) 通过超声处理将染色质剪切成小片段，(3) 目标蛋白的免疫沉淀，(4) 逆转交联，以及 (5) 通过 PCR 定量特定的相关 DNA 序列。在这里，我们介绍并讨论多年来我们开发的使用爪蟾细胞和蝌蚪组织作为实验材料进行 ChIP 测定的方案。
Understanding the accurate temporal and spatial regulation of gene expression during development requires knowledge of the spectrum of transcription factors and cofactors involved and their functional interplay with chromatin. Chromatin immunoprecipitation (ChIP) has become a powerful technique that allows us to do so. A typical ChIP assay involves (1) treating cells or tissues with formaldehyde to rapidly crosslink chromatin-associated proteins to DNA, (2) shearing chromatin by sonication into small fragments, (3) immunoprecipitation of the proteins of interest, (4) reversal of crosslinking, and (5) quantitating the specific associated DNA sequences by PCR. Here we present and discuss the protocols we have developed over the years for ChIP assays usingXenopusoocytes and tadpole tissues as experimental materials.