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Browsing by Author "Kuan Kiat, Chew"

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  • Kuan Kiat, Chew (2019)
    Proteostasis is used by cells to maintain proteome health and understanding the biological mechanisms underpinning proteostasis is important. Despite many studies on small molecule-mediated inhibition of Sec61-dependent protein translocation, a knowledge gap exists in the quality control pathway(s) of pharmacologically-displaced secretory polypeptides. Genetic screens can be used to discover proteostasis regulators of pharmacologically-displaced secretory polypeptides. Near-haploid human HAP1 cells with an inducible Tet-on GFP reporter (reporter HAP1 cells) can be used as a cellular tool to screen for human host factors pertinent to proteostasis of secretory polypeptides. The isolation of haploid-enriched reporter HAP1 cells ensures that the inability to efficiently recover bi-allelic gene trap mutants is avoided. The use of haploid-enriched cells is a prerequisite to gene trap mutagenesis screens. Here, I present data on the isolation of haploid-enriched reporter HAP1 cells that could be used as a cellular tool in gene trap mutagenesis screens. A workflow for the isolation of haploid-enriched reporter HAP1 cells was optimised using a diploid reporter HAP1 cell line as a control. Both DNA content analysis and karyotyping showed that the isolated HAP1 reporter cell lines were haploid. In the haploid-enriched HAP1 reporter cells, the GFP-reporter compartmentalised in the ER, and a Sec61-translocon inhibitor CT8 could inhibit the GFP-mediated fluorescence. The haploid reporter HAP1 cell lines produced in this study are suitable for future gene-trap mutagenesis experiments.