Fluorescence In Situ Hybridization on 3D Cultures of Tumor Cells
Genomes are spatially highly organized within interphase nuclei. Spatial genome organization is increasingly linked to genome function. Fluorescence in situ hybridization (FISH) allows the visualization of specific regions of the genome for spatial mapping. While most gene localization studies have been performed on cultured cells, genome organization is likely to be different in the context of tissues. Three-dimensional (3D) culture model systems provide a powerful tool to study the contribution of tissue organization to gene expression and organization. However, FISH on 3D cultures is technically more challenging than on monocultures. Here, we describe an optimized protocol for interphase DNA FISH on 3D cultures of the breast epithelial cell line MCF-10A.B2, which forms breast acini and can be used as a model for early breast cancer.
Key wordsFISH 3D cell culture Acini Interphase Nuclear architecture Tissue models Genome organization Early tumorigenesis
The author would like to thank Dr. T. Misteli for critical reading of this chapter and for his continued advice and encouragement; Dr S. Muthuswamy CSHL, NY, USA) for providing the MCF10.B2 cell line; and ARIAD Pharmaceuticals (http://www.ariad.com/regulationkits) for providing the synthetic ligand AP1510. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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