Meadowswaters0742

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4+ T cells from healthy donors, or cells infected in vitro with the virus. Liraglutide price Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.A comprehensive analysis of the tridimensional (3D) organization of the genome is crucial to understand gene regulation. Three-dimensional DNA fluorescent in situ hybridization (3D-FISH) is a method of choice to study nuclear organization at the single-cell level. The labeling of DNA loci of interest provides information on their spatial arrangement, such as their location within the nucleus or their relative positioning. The single-cell information of spatial positioning of genomic loci can thus be integrated with functional genomic and epigenomic features, such as gene activity, epigenetic states, or cell population averaged chromatin interaction profiles obtained using chromosome conformation capture methods. Moreover, the development of a diversity of super-resolution (SR) microscopy techniques now allows the study of structural chromatin properties at subdiffraction resolution, making a finer characterization of shapes and volumes possible, as well as allowing the analysis of quantitative intermingling of genomic regions of interest. Here, we present and describe a 3D-FISH protocol adapted for both conventional and SR microscopy such as 3D structured illumination microscopy (3D-SIM), which can be used for the measurement of 3D distances between loci and the analysis of higher-order chromatin structures in cultured Drosophila and mammalian cells.The chromosomes in mammalian interphase nuclei are organized into domains called chromosome territories that play a major role in nuclear organization. Here we propose a methodology that combines the use of micro-patterning of adhesive molecules to impose single-cell geometry, with visualization of chromosome territories. This allows obtaining a representative statistical map of the absolute positions of chromosome territories relative to the geometry imposed to the cell population by combining the signal from each cell.The organization of the eukaryotic nucleus facilitates functional chromatin contacts which regulate gene transcription. Despite this being extensively studied through population-based chromatin contact mapping and microscopic observations in single cells, the spatiotemporal dynamics of chromatin behavior have largely remained elusive. The current methods to label and observe specific endogenous genomic loci in living cells have been challenging to implement and too invasive to biological processes. In this protocol, we describe the use of a recently developed DNA labelling strategy (ANCHOR) with CRISPR/Cas9 gene editing, to discreetly label genes for live cell imaging to study chromatin dynamics. Our approach improves on some of the fundamental shortfalls associated with current labelling strategies and has the potential for multiplexed observations.Genome architecture and function are strictly related to nuclear structures, which contact chromatin at specific regions, regulating its compaction and three-dimensional higher-order structure, therefore contributing to specialized gene expression programs. Recently, growing evidence uncovers a dynamic role of nuclear structures in the plasticity of transcriptional programs. When the cellular microenvironment changes, external cues are transmitted to the nucleus through complex signalling cascades, finally resulting in a genome reorganization that allows the adjustment of the cell to a new condition. This process can be very rapid, especially in cells whose function is to contain sudden threats to the organism. Some examples are stem cells that switch from a quiescent to an activated state to replace damaged tissues or immune cells that, with a similar dynamic, identify and eliminate pathogens.Experimental treatments often require the isolation of cells from their physiological environment, exposing them to possible sudden changes in their nuclear architecture. Here we propose an early cross-linking on primary cells, a fixing method that can help to minimize the risk of nuclear structure alteration during the isolation process. We also bring some examples of downstream studies on early-fixed cells.The organization of DNA within the eukaryotic nucleus is important for cellular processes such as regulation of gene expression and repair of DNA damage. To comprehend cell-to-cell variation within a complex system, systematic analysis of individual cells is necessary. While many tools exist to capture DNA conformation and chromatin context, these methods generally require large populations of cells for sufficient output. Here we describe single-cell DamID, a technique to capture contacts between DNA and a given protein of interest. By fusing the bacterial methyltransferase Dam to nuclear lamina protein lamin B1, genomic regions in contact with the nuclear periphery can be mapped. Single-cell DamID generates contact maps with sufficient throughput and resolution to reliably identify patterns of similarity as well as variation in nuclear organization of interphase chromosomes.