Reverse genetic analysis of Cep164 in vertebrates
Daly, Owen
Daly, Owen
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Publication Date
2016-05-12
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Thesis
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Abstract
Components of the centrosome have been reported to have a role in the DNA Damage Response (DDR) pathway. Cep164, a protein localised to the distal appendages of centrioles, is theorised to play a part in the DDR. As with the centrosomal protein Centrin2, Cep164 has been linked to Nucleotide Excision Repair (NER), the mechanism used to repair DNA lesions induced by UV light. This link has been established through a study which reported that Cep164 interacts with the NER repair factor XPA and that Cep164 depletion results in hampered survival after UV-induced DNA damage. To further elucidate the centrosomal and DNA repair roles of Cep164, we targeted its gene locus in both chicken and human model systems. Our targeting strategy in chicken DT40 cells was designed to attach both an Auxin Inducible Degron (AID) and a GFP tag to the Cep164 protein. This allowed us to track and deplete the protein in a controlled manner. In the human RPE-1 cell-line, we conducted a gene disruption of CEP164 using CRISPR/Cas9. We verified the successful gene targeting in both cell lines through a combination of Western and/or Southern analysis. Using immunofluorescence microscopy we confirmed the localisation of the Cep164 protein to the centrosome and also described a novel monoclonal antibody to human Cep164. This is the first study examining a Cep164 knockout cell line. Our results indicate that Cep164 is essential for ciliogenesis. Through transmission electron microscopy analysis of the human knockout we confirm that Cep164 is necessary for the binding of distal appendage vesicles to the mother centriole. However, in contrast to published data, we have shown Cep164 to be dispensable for the DNA damage response. We show exclusive localisation of Cep164 to the centrosome after DNA damage and through clonogenic survival assays, have found no sensitivity to IR or UV radiation when Cep164 is absent.
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Attribution-NonCommercial-NoDerivs 3.0 Ireland