Person: Knouse, Kristin
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Knouse
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Kristin
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Knouse, Kristin
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Publication Detection of Copy Number Alterations Using Single Cell Sequencing(MyJove Corporation, 2017) Knouse, Kristin; Wu, Jie; Hendricks, AustinDetection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.Publication Tissue Architecture Is Required for Chromosome Segregation Fidelity in Epithelia(2018-05-15) Knouse, KristinChromosome segregation is classically viewed as a cell-intrinsic process. We tested this assumption by comparing chromosome segregation fidelity in various epithelial cells in their native tissue and as dissociated cells in culture and discovered that chromosome segregation fidelity is dependent on the tissue environment. Using organoid culture systems, we show that it is tissue architecture, the cell adhesion and cell polarity patterns that define a tissue, which is responsible for the heightened chromosome segregation fidelity in tissues and that disruption of tissue architecture leads to chromosome instability. Tissue architecture enhances the cell’s ability to correct erroneous microtubule-kinetochore attachments and this enhancement is especially important for maintaining chromosome stability in the liver, a polyploid tissue with high levels of erroneous attachments. Moreover, we find that neural progenitor cells, which proliferate in a region of the brain that lacks defined architecture, maintain their polarity and chromosome segregation fidelity even when removed from their tissue environment. We show that disruption of cell polarity in epithelia recapitulates the chromosome segregation defects observed in dissociated cells, arguing that cell polarity is the critical component of tissue architecture that facilitates chromosome segregation. Our data bring us to the surprising conclusion that the external environment influences chromosome segregation across many epithelial cell types. The function of epithelial tissues requires that cells maintain distinct adhesions and polarity in both interphase and mitosis. In relinquishing autonomy over their polarity, epithelial cells also rely on their native tissue for chromosome segregation fidelity. We propose that disruption of tissue architecture could explain the chromosome instability that characterizes and drives cancer. More broadly, our observations highlight the importance of context for even the most fundamental cellular processes and thus the need to use experimental systems that maximize physiologic relevance across all areas of cell biology.