Core 1: Mouse and Cell Modeling

核心 1：小鼠和细胞建模

• 批准号: 10599915
• 负责人: Bing Xia
• 金额: $ 25.09万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599915
• 关键词: Address; Alleles; BARD1 gene; BRCA1 gene; BRCA2 gene; Binding; Biological Process; Breast; Breast Cancer Cell; Breast Cancer cell line; Breeding; CRISPR/Cas technology; Cell Line; Cell model; Cells; Chemoresistance; Chromosome 11; Chromosomes; Complex; Custom; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; Dedications; Defect; Development; Dissection; ES Cell Line; Embryo; Ensure; Evaluation; Fertilization in Vitro; Fibroblasts; Future; Generations; Genes; Genetic; Genetically Engineered Mouse; Genomics; Guidelines; Human; Human Cell Line; Individual; Institution; Knock-out; Knockout Mice; Laboratories; LoxP-flanked allele; MCM10 gene; Malignant Neoplasms; Modeling; Modification; Mus; Mutation; Organism; PALB2 gene; Process; Production; Proteins; Reagent; Regulation; Research; Research Personnel; Site; Structure; Structure-Activity Relationship; Technology; Testing; base editing; cancer prevention; cancer therapy; cell type; design; embryonic stem cell; genetically modified cells; genome editing; germ free condition; inhibitor; medulloblastoma; medulloblastoma cell line; mouse model; novel; p53-binding protein 1; preservation; protein protein interaction; rational design; recruit; replication stress; tool; treatment response; triple-negative invasive breast carcinoma; tumor progression; tumorigenesis

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.

项目摘要（核心 1） 基因工程细胞系和小鼠模型对于阐明复杂的生物过程至关重要。 定制提出这些科学问题的背景的能力达到了前所未有的水平 细节和技术精度。本质上，任何遗传目标都可以改变以解决蛋白质定位问题， 内源表达水平的蛋白质-蛋白质相互作用或结构-功能关系 规定。小鼠和细胞建模核心（核心 1）将为 P01 项目创建新的研究材料 并重新利用多个已建立的小鼠模型，以原代细胞系的方式产生新的试剂。 借助基于 CRISPR/Cas9 的基因编辑工具，Core 1 将修改人类细胞系，从而能够解剖 各个 P01 项目中描述的 BRCA 网络。乳腺癌中的关键基因将被破坏 髓母细胞瘤细胞系和参与 DNA 修复和复制应激缓解的蛋白质将被修改 扰乱他们的互动领域。研究人员之间的这种综合方法将允许交流 与每个项目相关的结果并告知未来的方向。小鼠胚胎成纤维细胞 (MEF) 和 将产生胚胎干 (ES) 细胞系来研究 DNA 损伤位点的蛋白质定位和 除了所需的突变外，修复效率接近野生型背景。在生产所需的 MEF、基因匹配的对照将同时产生。常用的细胞系可能有 DNA 修复途径中未表征的缺陷，而新生成的 MEF 和 ES 细胞接近天然 状态。将通过向现有 Brca1/Trp53 条件系添加空等位基因来制作独特的小鼠模型 化学耐药性的强预测因子。由于 Brca1、Trp53 和新靶点均位于同一位置 染色体，CRISPR/Cas9将被用于体外受精（IVF）衍生的胚胎中以破坏该基因 无需进行广泛的杂交来结合三个物理上接近的等位基因。另一种独特的 小鼠模型将通过删除 Brca1 中编码关键结合区的基因组序列来制作 互动伙伴。核心 1 借鉴了小鼠胚胎处理、小鼠胚胎成纤维细胞和 胚胎干细胞生产、细胞系基因编辑、细胞系敲除评估、小鼠模型创建、 CRISPR/Cas9项目设计、复杂的项目管理和现有小鼠品系的高效育种。经过 在核心1中集中生产基因编辑细胞系，可以集中于各个实验室的研究 关于下游分析。