Pradhan Roopali, Sengupta Kundan
Biology, Main Building, Indian Institute of Science Education and Research (IISER), Pune, Maharashtra, India.
Methods Mol Biol. 2019;2038:181-197. doi: 10.1007/978-1-4939-9674-2_12.
It is well established that the genome is non-randomly organized in the interphase nucleus with gene rich chromosome territories toward the nuclear interior, while gene poor chromosome territories are proximal to the nuclear periphery. In vivo tissue stiffness and architecture modulates cell type-specific genome organization and gene expression programs. However, the impact of external mechanical forces on the non-random organization of the genome is not completely understood. Here we describe a modified protocol for visualizing chromosome territories and gene loci positions in cells exposed to reduced matrix stiffness by employing soft polyacrylamide matrices. 3-Dimensional Fluorescence In Situ Hybridization (3D-FISH) protocol followed by image analyses performed on cells exposed to extracellular matrices of varying stiffness properties, enables the determination of the dynamics of chromosome territories as well as gene loci in the interphase nucleus. This will be useful in understanding how chromosome territories respond to changes in substrate stiffness and the potential correlation between the repositioning of chromosome territories and their respective transcriptional profiles.
众所周知，基因组在间期核中呈非随机组织，富含基因的染色体区域朝向核内部，而基因贫乏的染色体区域靠近核周边。体内组织硬度和结构调节细胞类型特异性的基因组组织和基因表达程序。然而，外部机械力对基因组非随机组织的影响尚未完全了解。在此，我们描述了一种改进的方案，通过使用柔软的聚丙烯酰胺基质来可视化暴露于降低的基质硬度的细胞中的染色体区域和基因座位置。对暴露于具有不同硬度特性的细胞外基质的细胞进行三维荧光原位杂交（3D-FISH）方案，然后进行图像分析，能够确定间期核中染色体区域以及基因座的动态变化。这将有助于理解染色体区域如何响应底物硬度的变化以及染色体区域重新定位与其各自转录谱之间的潜在相关性。
