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Wsb1 and Ksr1 gene add-backs to Chaos3 mammary tumor cells to analyze their role

Author
Shah, Hina
Abstract
The RAS protein is crucial in signaling pathways that regulate cellular proliferation. Tumors can form when this normal growth regulation breaks down due to defects in signaling mechanisms. The Chaos3 mouse model has a point mutation in the Mcm4 gene, a DNA replication licensing factor, which triggers replicative stress and genome instability, ultimately resulting in tumor formation. It was previously seen that two genes, Wsb1, a E3 ubiquitin ligase, and Ksr1, a kinase suppressor of RAS, were commonly deleted in Chaos3 tumors. In this project, I used mammary tumor cells from the Chaos3 mouse model to study the effect of re-introducing RAS-regulatory factors. In order to accomplish this, stable Chaos3 mammary tumor cell lines were created with the random integration of ectopic transgenes (complementations) that corresponded to endogenous genes deleted in these cells. I confirmed successful insertions through RT-qPCR, immunofluorescence, and western blotting. I also analyzed DNA replication (through EdU labeling), growth curves, and the amount of active RAS. I found that KSR1 and a fragment of WSB1 both resulted in fewer replicating cells, but had different effects on RAS. Future research should involve replicating these preliminary experiments to elucidate potential involvement of the Ksr1 and Wsb1 genes in mammary tumor formation.
Date Issued
2019-05Subject
Biological sciences honors program; tumorigenesis; WSB1; KSR1; cell growth; mammary tumor; chaos3; mouse model
Degree Discipline
Biological Sciences
Degree Name
B.A., Biological Sciences
Degree Level
Bachelor of Arts
Type
dissertation or thesis