Date of Award
© 2021 Andrew Y. Cheng. All rights reserved. This work is freely available courtesy of the author. It may only be used for non-commercial, educational, and research purposes. For all other uses, including reproduction and distribution, please contact the copyright holder.
Bachelor of Arts
Dawn M. Carone
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.
Cheng, Andrew Y. , '21, "Sharpening new scissors: Designing a CRISPR-Cas9 construct for inducible and interchangeable HSATII expression in Tig-1 fibroblasts" (2021). Senior Theses, Projects, and Awards. 166.