Core 1: Mouse and Cell Modeling

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PROJECT SUMMARY (Core-1) Genetically engineered cell lines and mouse models are critical for elucidating complex biological processes. The ability to customize the context in which these scientific questions are asked is at an unprecedented level of detail and technical precision. Essentially, any genetic target can be altered to address protein localization, protein-protein interaction or structure-function relationships at endogenous levels of expression and regulation. The Mouse and Cell Modeling Core (Core 1) will create new research materials for the P01 projects and repurpose multiple, established mouse models to generate novel reagents in the way of primary cell lines. With CRISPR/Cas9-based gene editing tools, Core 1 will modify human cell lines to enable the dissection of the BRCA network as described in the individual P01 projects. Key genes will be disrupted in breast cancer and medulloblastoma cell lines and proteins involved in DNA repair and replication stress relief will be modified to disrupt their interaction domains. This comprehensive approach among the investigators will allow exchange of results related to each the projects and inform future directions. Mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cell lines will be generated to study protein localization to sites of DNA damage and repair efficiency in near wild-type backgrounds, except for the desired mutations. In producing the desired MEFs, genetically matched controls will be produced concomitantly. Commonly used cell lines may have uncharacterized defects in DNA repair pathways, while newly generated MEFs and ES cells are near native state. A unique mouse model will be made by adding to an existing Brca1/Trp53 conditional line a null allele for a strong predicator of chemoresistance. As Brca1, Trp53 and the new target are all located on the same chromosome, CRISPR/Cas9 will be used in embryos derived by in vitro fertilization (IVF) to disrupt the gene without having to do extensive interbreeding to combine the three physically close alleles. Another unique mouse model will be made by deleting the genomic sequence in Brca1 that encodes the binding region of key interaction partner. Core 1 draws on expertise in mouse embryo handling, mouse embryonic fibroblast and embryonic stem cell production, cell line gene editing, evaluation of cell line knock-outs, mouse model creation, CRISPR/Cas9 project design, complex project management and efficient breeding of existing mouse lines. By concentrating production of gene edited cell lines in Core 1, research in individual laboratories can be focused on downstream analyses.