Departmental Honors Project Title
Date of Award
College of Liberal Arts
Cancer, the second leading cause of death in the US, is caused by mutations in select genes that alter cellular function leading to uncontrolled proliferation. Understanding the specific genes that drive cancer can lead to the generation of novel cancer therapies. To identify novel genes that drive cancer in the colon (CRC), lungs, and ovaries in mice, Starr et al. employed a transposon-based insertional mutagenesis system. One of the genes identified, APC, is mutated in 70-80% of human CRCs. CUL3, suspected to be a general driver gene, was discovered in the lung cancer screen. CUL3 was analyzed for its role in a human CRC cell line in this study. CUL3 gene knockout was performed using the CRISPR/Cas9 system, which targets mutations to specific genes, thereby knocking out that gene’s function. Three different sites in the CUL3 gene were targeted for mutation and resulted in the creation of 41 separate cell lines with potential CUL3 knockout. Of those 41 cell lines, 25 exhibited qualitatively abnormal phenotypes 10 days after transfection. These phenotypes include slowed growth (25 of 25 cell lines), increased cell size (16 of 25 cell lines), and variation of cell adherence to culture flask surface (11 of 25 cell lines). Knockout was confirmed in 6 cell lines by using PCR in the region of the gene targeted for mutation and sequencing the PCR product. Each cell line was quantitatively evaluated for metabolic activity (or cell growth rate) using an MTS assay. If CUL3 knockout is shown to reduce overall cell growth and increase susceptibility to chemotherapy, this would support the development of new therapies for CRCs that target CUL3 function.
Lautz, Zoe A., "The Significance of CRISPR/Cas9-Directed CUL3 Knockout on Human Colorectal Cancer Cells" (2015). Departmental Honors Projects. 40.
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