Date of Award
Master of Science (MS)
College of Science and Mathematics
Thesis Sponsor/Dissertation Chair/Project Chair
Inducible cAMP Early Repressor (ICER) is a dominant transcription repressor of the cAMP Responsive Element Modulator (CREM) and cAMP Responsive Element Protein (CREB) family of transcription factors. ICER isoforms are differently expressed in various cell types and are involved in essential functions including hormonal regulation, circadian rhythm, spermatogenesis, and immune response. In the context of cancer, ICER is proposed as a tumor suppressor in leukemia, prostate, pituitary cancers, and melanoma. In melanoma, ICER is targeted for degradation during tumorigenesis and highly upregulated during regression, further highlighting its potency as a tumor suppressor. Inside the nucleus, ICER targets oncogenes and key cell-cycle regulatory genes such as Cyclin A, Cyclin D, C-fos and Bcl2, yet the full spectrum of its targets has not been fully explored.
The goal of this thesis is to generate a stable inducible cell line which allows better study of ICER at viable conditions, identifying its binding regions and any additional genes it targets. For this study Sk-Mel-24 cells were used as a model of human melanoma. Two plasmids were utilized in each transfection round, all-in-one Cas9 gRNA plasmid and an ICER-containing donor DNA plasmid. The Cas9 gRNA plasmid was used to target the AAVS1 (safe harbor) locus, due to the transcriptional competence and minimal adverse effect of the region. As for the donor plasmid, it carried N-terminus hemagglutinin (HA) tagged ICER (NHA-ICER) under the control of Tetracycline-induced expression system. Results from Western blot, immunocytochemistry (ICC), fluorescence microscopy all demonstrate successful doxycycline induction. Furthermore, PCR and sanger data proper integration of NHA-ICER into the AAVS1 locus.
Hallak, Abdulkader, "Optimizing CRISPR/Cas9 Transfection Protocol to Generate a Tet-on Inducible Sk-Mel 24 Cell Lines to Better Study ICER" (2023). Theses, Dissertations and Culminating Projects. 1205.