Date of Award
Master of Science
Many physiological parameters such as body temperature, circulating hormone levels, blood pressure and metabolic activity are rhythmic, and their rhythmicity is governed by the circadian clock. This is a self-sustained ̃24-hour cycle, which at the molecular level is determined by negative transcriptional and translational feedback loops controlled by the products of core clock genes. There are at least 10 core clock genes namely Bmal1, Bmal2, Clock, Npas2, Per1, Per2, Per3, Cry1, Cry2, Nr1d1 and Nr1d2. In addition to these core clock genes, clock modifier genes exist that modulate the molecular clock by integrating synchronous and asynchronous clues from signal transduction pathways. Nearly 1000 clock modifier genes have been identified and among these is the chemotactic cytokine-like protein CKLF. shRNA mediated down-regulation of Cklf conducted in U2OS cell lines exhibited short period length, whereas its knockdown in MMH-D3 reporter cells resulted in decreased amplitude of oscillation, suggesting a role for Cklf in regulating the oscillatory function of the clock. Since Cklf is an immune mediator, this result suggests a functional interaction between the immune system and the clock. To further investigate the interaction between Cklf and the core clock genes and to determine how the circadian clock is integrated with and regulated by immune mediators, we carried out knockout studies using the latest Crispr/Cas9 gene editing tool. Crispr/Cas9 is an efficient genome editing tool that allows for genetic perturbation of individual genes with enhanced specificity and efficacy of gene knockout compared to RNAi approaches. Using Crispr/Cas9 method my work focused on knocking out Cklf and one of the core clock genes Cry2 in MMH-D3 reporter cell lines. We focused on Cry2 because it is one of the key proteins that controls the period of the clock. We successfully knocked out Cry2 and showed that the knockout cells exhibited long-period phenotype, confirming that Cry2 is core clock gene thatfunctions to maintain the period of the clock to about 24 hours. In contrast, repeated efforts to knock out Cklf using the Crispr/Cas9 approach was unsuccessful. Future work aims to obtain Cklf knockout cell lines by improving the genome editing efficiency such as higher transfection efficiency, expanding flow cytometry-based single cell sorting and cloning, and designing new PCR genotyping strategies.
Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses and dissertation (ETD) repository.
Ramanna, Thanuja, "Generation of knockout cell lines to investigate the roles of Cklf and Cry2 in the regulation of circadian clock" (2018). Electronic Theses and Dissertations. 2379.