Date of Award

5-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Carlos A. Molina

Committee Member

Kirsten Monsen-Collar

Committee Member

Sandra Adams

Abstract

Melanoma is one of the most aggressive forms of cancer and is prone to metastasis. Once melanoma metastasizes, it is difficult to treat as patients often become immune to MAPK inhibitors and other therapeutic agents. Small molecules, such as immune checkpoint inhibitors, have demonstrated success as an adjunct to melanoma treatment. As a small, 18kDa molecule with tumor suppressing properties, research using ICER continues to provide positive anti-tumor effects. ICER regulates cAMP-induced transcription as a powerful, dominant-negative transcriptional repressor. Recent research demonstrated that the fusion tag hemagglutinin (HA), when fused to ICER’s N-terminus, increased ICER’s half-life and apoptotic activity in SK-MEL-24 cells. If a stable, inducible HAN-ICER cell line is established, much more research will be able to take place to further understand the role of ICER in melanoma cells. Until now, a stable HAN-ICER cell line has not been developed despite many efforts to accomplish this in the laboratory. Here, it was demonstrated that co-transfection with a homologous recombination (HR) plasmid and a Cas9 gRNA plasmid which targets the AAVS1 safe harbor locus were used to incorporate EGFP into the genome of SK-MEL-24 cells as a proof of concept. Results were confirmed by fluorescent microscopy and Sanger sequencing data. Additionally, HAN-ICER was integrated into the SK-MEL-24 cellular genome, and results were confirmed by western blot and immunocytochemistry (ICC).

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