Mitotik kinaz fonksiyonlarinin kimyasal genetik yaklaşimlarla i̇ncelenmesi

Mitosis is the shortest phase of the cell cycle yet it involves dramatic changes in the cellular architecture and processes. Mitotic entry and progression are tightly regulated by the dynamic actions of signaling pathway networks and autoamplification loops. Mastl kinase is responsible for preservation of the hyperphosphorylated state of the mitotic substrates by indirectly inhibiting the major antagonist protein phosphatase, PP2A. Loss of function studies of Mastl kinase have shown defects of chromosomal segregation and subsequent failure of cytokinesis, and underphosphorylation of the mitotic substrates. To better understand and further characterize the mitotic functions of Mastl kinase, we aimed to engineer its analogue-sensitive mutant variants. We initially worked on editing the endogenous loci in human cell lines using CRISPR-Cas9 system. After failed attempts of editing the endogenous loci, we decided to use the conditional knockout mouse embryonic fibroblasts. Using retroviral vectors, we ectopically expressed the mutant variants of mouse Mastl and deleted the endogenous loci by inducing knockout in these stable cell lines respectively. Our eventual aim was to create an enzyme that will bind ATP and have normal kinase activity, and yet, be sensitive to an ATP analogue inhibitor. As a result, we observed that the stable cell lines are not viable when knockout was induced. We suspect the possible negative impacts of the mutations on enzymatic activity and insufficient ectopic expression levels. In order to compensate for this possible partial enzymatic inactivation, we are currently switching to lentiviral expression systems to achieve higher expression levels.