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dc.contributor.advisorSchwartz, Jacob C.en
dc.contributor.authorWhite, Connor Ian
dc.creatorWhite, Connor Ianen
dc.date.accessioned2017-08-10T21:12:48Z
dc.date.available2017-08-10T21:12:48Z
dc.date.issued2017
dc.identifier.urihttp://hdl.handle.net/10150/625242
dc.description.abstractThe FET proteins, FUS, EWSR1, and TAF15 are RNA binding proteins that form amyloid-like plaques through aggregation. Research into these proteins is important due to their presence and implication in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The objective of this project was to clone the FET genes into an in-vitro expression system. Using this method, the need to culture cells to obtain protein for in-vitro experiments testing protein-protein interactions and RNA binding ability would be removed. This project was based around the cloning of each FET gene into a pT7CFE1-CHis plasmid vector using MEGAWHOP protocols. After cloning of a gene, in-vitro translation (IVT) using HeLa lysates was facilitated using the internal ribosomal entry site (IRES) sequence found in the pT7CFE1-CHis plasmid. During this project, in-vitro translation constructs were designed and tested with a pilot in-vitro translation assay performed using the initial FUS in-vitro translation MEGAWHOP product, revealing the unexpected result of a double insertion of FUS into the pT7CFE1-CHis plasmid. After alterations to the in-vitro translation constructs, FUS and TAF15 megaprimers have been reliably produced for proper insertion into the pT7CFE1-CHis plasmid.
dc.language.isoen_USen
dc.publisherThe University of Arizona.en
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en
dc.titleIn-Vitro Expression of FET Proteinsen_US
dc.typetexten
dc.typeElectronic Thesisen
thesis.degree.grantorUniversity of Arizonaen
thesis.degree.levelbachelorsen
thesis.degree.disciplineHonors Collegeen
thesis.degree.disciplineBiochemistryen
thesis.degree.nameB.S.en
refterms.dateFOA2018-09-11T22:15:21Z
html.description.abstractThe FET proteins, FUS, EWSR1, and TAF15 are RNA binding proteins that form amyloid-like plaques through aggregation. Research into these proteins is important due to their presence and implication in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The objective of this project was to clone the FET genes into an in-vitro expression system. Using this method, the need to culture cells to obtain protein for in-vitro experiments testing protein-protein interactions and RNA binding ability would be removed. This project was based around the cloning of each FET gene into a pT7CFE1-CHis plasmid vector using MEGAWHOP protocols. After cloning of a gene, in-vitro translation (IVT) using HeLa lysates was facilitated using the internal ribosomal entry site (IRES) sequence found in the pT7CFE1-CHis plasmid. During this project, in-vitro translation constructs were designed and tested with a pilot in-vitro translation assay performed using the initial FUS in-vitro translation MEGAWHOP product, revealing the unexpected result of a double insertion of FUS into the pT7CFE1-CHis plasmid. After alterations to the in-vitro translation constructs, FUS and TAF15 megaprimers have been reliably produced for proper insertion into the pT7CFE1-CHis plasmid.


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