This project will investigate mammalian DNA polymerase ? (Pol ?), the defining enzyme for repair of DNA double-strand breaks by polymerase theta-mediated end joining (TMEJ). This is Core C (?Cell Line Engineering Core?) which is part of a Program Project titled, ?Polymerase theta, genome instability, and cancer?. Core C will be responsible for providing native and genetically engineered cell lines to support all of the cell-based assays performed in Projects 1, 2, and 4 of the Program Project. The authentication of mammalian cell line reagents is vital for rigor and reproducibility of laboratory experiments. By establishing and implementing standardized protocols for cell line validation, efficient genome manipulation, cryopreservation, and dissemination, the Core will ensure quality control of cell line reagents and minimize the likelihood of erroneous observations and interpretations.
In Aim 1, ?Credentialing of mammalian cell line models utilized in this Program Project?, we will implement a standardized and comprehensive protocol for authentication and characterization of mammalian cell line models used across the Program Project.
In Aim 2, ?CRISPR-based cell line genome engineering services for the Program Project?, we will provide expert services in the design and execution of customized cell line genome engineering tasks in support of Program Project goals. Cas9 protein utilized in this Aim will be provided by Core B.
In Aim 3, ?Controlled and stable expression of transgenes in mammalian cell lines?, we will utilize doxycycline-inducible and constitutive transposon vectors for the generation of cell lines that express a variety of mutagenized and/or tagged transgenes of interest. Core C services will be highly coordinated within the Program Project, and will facilitate rigor and reproducibility, as well as cost-effectiveness, of highly integrated research activities aimed at understanding the role and targetability of DNA Polymerase q in cancer.
This project will investigate mammalian DNA polymerase theta, the defining enzyme for a biologically important mechanism for repair of genomic damage. This Core will generate and validate mammalian cell line reagents that are vital for a multidisciplinary program project that will uncover vulnerabilities that arise when this repair process is defective. This research is directly relevant to the future of successful polymerase theta-based therapies.