A cellular tool for discovery of quality control factors for displaced secretory polypeptides: Development of a haploid human reporter cell line expressing a secreted GFP reporter

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http://urn.fi/URN:NBN:fi:hulib-201906052331
Title: A cellular tool for discovery of quality control factors for displaced secretory polypeptides: Development of a haploid human reporter cell line expressing a secreted GFP reporter
Author: Kuan Kiat, Chew
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Faculty of Biological and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2019
URI: http://urn.fi/URN:NBN:fi:hulib-201906052331
http://hdl.handle.net/10138/302588
Thesis level: master's thesis
Abstract: 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.
Subject: Cellular tool
Genetic screen
Haploid-enriched reporter HAP1 cells
DNA content analysis
karyotyping
displaced secretory polypeptides
Discipline: biokemia
Biochemistry
biokemi
Full text embargoed until: 2019-10-04


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