Imaging Chromosome Territory and Gene Loci Positions in Cells Grown on Soft Matrices
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.
Key words3D-FISH Polyacrylamide matrices Stiffness Chromosome territories Gene loci
This work is supported by IISER Pune and Wellcome Trust–Department of Biotechnology India Alliance (Grant number: 500164/Z/09/Z) by funding through an intermediate fellowship to K.S. Council of Scientific and Industrial Research, New Delhi supported RP by Senior Research Fellowship. We gratefully acknowledge facilities and equipment of Indian Institute of Science Education and Research (IISER), Pune. We thank the IISER Pune Microscopy Facility and Chromosome Biology Lab (CBL) members for their comments and suggestions.
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