Cell Biology of Genomes Group, National Cancer Institute, NIH, Bethesda, MD 20892, United States.
NCI High-throughput Imaging Facility, National Cancer Institute, NIH, Bethesda, MD 20892, United States.
Methods. 2018 Jun 1;142:30-38. doi: 10.1016/j.ymeth.2018.01.013. Epub 2018 Feb 10.
The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.
染色体在核空间中的空间组织是一个广泛研究的领域，该领域依赖于对染色体的结构特征和 3D 位置进行高精度和高稳健性的测量。然而，目前尚无工具可用于以高通量格式对染色体结构域进行成像和分析。在这里，我们开发了高通量染色体结构域映射（HiCTMap）方法，该方法可用于稳健、快速地分析哺乳动物细胞中的 2D 和 3D 染色体结构域定位。HiCTMap 是一种基于高通量成像的染色体检测方法，可实现对染色体结构和核位置的常规分析。该方法使用优化的 FISH 染色方案在 384 孔板格式中结合定制的自动化图像分析工作流程，可在每个实验条件下对大量细胞中的染色体结构域及其在 2D 和 3D 中的位置进行忠实检测。我们将这项新技术应用于可视化男性和女性原代人皮肤成纤维细胞中的染色体 18、X 和 Y，并显示出在各自基因型中准确检测到正确数量的染色体。鉴于能够可视化和定量分析大量细胞核，我们使用 HiCTMap 以高精度测量染色体结构域的面积和体积，并使用质心或等距壳分析确定染色体结构域的径向位置。HiCTMap 方案还与 RNA FISH 兼容，如在雌性细胞中同时标记 X 染色体和 Xist RNA 所示。我们建议 HiCTMap 将成为在广泛的细胞类型和组织中常规精确映射染色体结构域的有用工具。
