Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35 kb-300 kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains. More specifically, we show that mapping of specific chromatin landscapes informs on changes associated with estrogen stimulated gene activity in human breast cancer cell lines.

中文翻译：

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3D FISH 分析人类癌细胞系中基因域特异性染色质重塑

荧光原位杂交 (FISH) 是一种常用技术，用于标记 DNA 和/或 RNA 以检测感兴趣的基因组区域。然而，该技术可能具有挑战性，特别是当应用于人类癌细胞中的单个基因时。在这里，我们提供了一个分步协议，用于在三个维度 (3D) 中分析短 (35 kb-300 kb) 基因组区域。我们讨论了实验设计，并为 3D 成像和数据分析提供了实际考虑，以确定染色质折叠。我们证明使用 BAC（细菌人工染色体）或 fosmids 的 3D FISH 可以提供基因域结构的详细信息。更具体地说，我们表明特定染色质景观的映射可以告知与人类乳腺癌细胞系中雌激素刺激的基因活性相关的变化。