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Sharpening new scissors: Designing a CRISPR-Cas9 construct for inducible and interchangeable HSATII expression in Tig-1 fibroblasts

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dc.contributor.advisor Carone, Dawn
dc.contributor.author Cheng, Andi Y.
dc.date.accessioned 2021-06-08T20:42:01Z
dc.date.available 2021-06-08T20:42:01Z
dc.date.issued 2021
dc.identifier.uri http://hdl.handle.net/10066/23029
dc.description.abstract As research delves ever deeper into the causes and progression of cancers, complex relationships between noncoding RNAs (ncRNAs) and tumorigenesis have been revealed. One such RNA is human satellite II (HSATII). While HSATII exists across multiple human chromosomes and comprises large portions of pericentromeric heterochromatin, it is transcriptionally silent in healthy cells. However, HSATII expression is aberrantly upregulated in many tumors, suggesting an etiological or exacerbatory role for the RNA transcripts. In cancerous cells, many aspects of cellular function are dysregulated, complicating the assessment of the specific effects of HSATII RNA. To investigate the role of HSATII RNA expression in cancer progression, a constitutive expression construct was previously designed for insertion into healthy Tig-1 fibroblast cells, generating cell lines that stably express HSATII over multiple generations. This project refines stable line generation and expression behavior by redesigning the expression construct to incorporate three new functionalities: 1) leveraging CRISPR-Cas9 editing enables targeted, sequence-specific genomic integration, 2) Golden Gate cloning facilitates the insertion of different/multiple HSATII sequence variants, and 3) a Tet-on promoter system produces inducible and controlled temporal expression of HSATII. An intermediary plasmid containing RFP as a marker for successful cloning was designed to expedite and simplify Golden Gate cloning. In the future, stable lines generated with this novel construct have the potential to more accurately parallel endogenous HSATII expression in cancers and offer high sequential, spatial, and temporal resolution for interrogating the role of HSATII ncRNA in tumorigenesis. en_US
dc.description.sponsorship Swarthmore College. Dept. of Biology en_US
dc.language.iso en en_US
dc.rights Full copyright to this work is retained by the student author. It may only be used for non-commercial, research, and educational purposes. All other uses are restricted.
dc.title Sharpening new scissors: Designing a CRISPR-Cas9 construct for inducible and interchangeable HSATII expression in Tig-1 fibroblasts en_US
dc.rights.access No restrictions en_US


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