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dc.contributor.advisor Segatori, Laura
dc.contributor.advisor Zhong, Weiwei
dc.creatorLiu, Yiwen
dc.date.accessioned 2017-08-02T14:27:16Z
dc.date.available 2017-08-02T14:27:16Z
dc.date.created 2017-05
dc.date.issued 2017-04-21
dc.date.submitted May 2017
dc.identifier.citation Liu, Yiwen. "Optimization of Proteasomal Degradation Reporter (eDeg-On) System for CRISPR-mediated Whole-genome Knockout Screens." (2017) Master’s Thesis, Rice University. https://hdl.handle.net/1911/96138.
dc.identifier.urihttps://hdl.handle.net/1911/96138
dc.description.abstract Protein folding and clearance of misfolded proteins are crucial to maintain cellular homeostasis (Jariel-Encontre et al., 2008). Misfolded proteins may associate with other cellular components and possibly impair their functions. They may also self-associate to form insoluble aggregates, which are the hallmarks of a number of neurodegenerative diseases, such as Parkinson’s (Olanow and McNaught, 2006) and Alzheimer’s (Oddo, 2008). The ubiquitin proteasome system (UPS) is the main pathway that catalyzes the degradation of soluble misfolded proteins in mammalian cells. Therefore, enhancing the UPS activity through activation of proteasomal degradation is considered a promising strategy to ameliorate phenotypes associated with the accumulation of misfolded proteins. Modulation of specific UPS components, for instance, results in increased degradation of target proteins (Rechsteiner and Hill, 2005 and Vilchez et al., 2012). However, our current understanding of the molecular mechanism underlying proteasomal degradation is still limited, limiting the rational design of pharmacologic strategies to enhance UPS activity. As a result, proteasome activators are rare and remain poorly characterized (Huang and Chen, 2009). To overcome these limitations, researchers in my group developed a cell-based platform (the eDeg-On system) to monitor changes in UPS activity. This genetic circuit links increase in UPS activity to an increase in fluorescent output, thereby providing a reliable tool for the discovery of proteasome activators. The CRISPR-cas technology has emerged as powerful technique to introduce genetic modifications at the whole-genome scale. I optimized the eDeg-On system and evaluated it for pooled screening of whole-genome CRISPR-mediated knockout library. I replaced the antibiotic resistance gene in the eDeg-On system and assessed the response of HEK293 cells stably expressing the eDeg-On system to modulation of proteasomal degradation. To evaluate the use of a stable cell line expressing the eDeg-On system as a reporter assay in the context of a pooled CRISPR-mediated screen, I conducted mock screens using different ratios of positive and negative controls. The results obtained demonstrate that the eDeg-On system can be used as a reporter assay for CRISPR-mediated whole-genome knockout screens. The use of the eDeg-On system to conduct genetic screen for the discovery of molecules that function as proteasome regulators will contribute to the development of therapeutic strategies for protein misfolding diseases. Further applications of this study include targeting the UPS function for therapeutic applications as well as for enhancing the production of recombinant proteins in industrial settings.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subjectUPS
CRISPR-cas
eDeg-On
protein degradation
dc.title Optimization of Proteasomal Degradation Reporter (eDeg-On) System for CRISPR-mediated Whole-genome Knockout Screens
dc.date.updated 2017-08-02T14:27:16Z
dc.type.genre Thesis
dc.type.material Text
thesis.degree.department Biochemistry and Cell Biology
thesis.degree.discipline Natural Sciences
thesis.degree.grantor Rice University
thesis.degree.level Masters
thesis.degree.name Master of Arts
thesis.degree.major Molecular Biology


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