High-throughput targeted mutagenesis of mouse stem cell lines

小鼠干细胞系的高通量定向诱变

• 批准号: 7687011
• 负责人: Pieter J. de Jong
• 金额: $ 468.95万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-07 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7687011
• 关键词: 129 Mouse; Alleles; Archives; Bacteria; Bacterial Artificial Chromosomes; Biology; Biomedical Research; Blood capillaries; C57BL/6 Mouse; California; Cataloging; Catalogs; Cell Line; Characteristics; Chimera organism; Collection; Communication; Communities; Core Facility; Cryopreservation; DNA; Data Coordinating Center; Databases; Detection; Development; Dideoxy Chain Termination DNA Sequencing; ES Cell Line; Electroporation; Embryo; Ensure; Environment; Exons; Feedback; Freezing; Frequencies; Funding; Gene Mutation; Gene Targeting; Generations; Genes; Genetic; Genetic Engineering; Genomics; Genotype; Germ Lines; Goals; Growth; Human; In Vitro; Institutes; Introns; Karyotype; Knock-out; Laboratories; LacZ Genes; Libraries; Life; Maps; Modeling; Modification; Monitor; Mouse Strains; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Needs Assessment; Operative Surgical Procedures; Pediatric Hospitals; Performance; Phenotype; Process; Production; Public Domains; Quality Control; Reagent; Reporter; Reporter Genes; Research; Research Infrastructure; Research Institute; Research Personnel; Resources; Robotics; Site; Specific qualifier value; Stem cells; Structure; Techniques; Technology; Testing; Trust; United States National Institutes of Health; Universities; Validation; Work; base; capillary; design; design and construction; embryonic stem cell; expectation; experience; feeding; homologous recombination; improved; in vivo; large scale production; member; mouse genome; mutant; null mutation; parallel processing; pathogen; pluripotency; programs; quality assurance; research study; sperm cell; technological innovation; transmission process; vector

DESCRIPTION: (provided by applicant) We propose the creation of 10,000 knock-out alleles of mouse genes exclusively by gene targeting. The generation of mutant mice from C57BL/6-derived embryonic stem cells is currently not sufficiently robust for this project. Therefore we will start with ES cells derived from a highly-efficient 129 mouse strain, switching to C57BL/6 ES cells as soon as they have been validated for high-throughput gene targeting. The work will be done by a three member consortium of the Children's Hospital Oakland Research Institute in Oakland, CA (CHORI), the Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK and the University of California Davis Mouse Biology Program (UCD-MBP). Targeting vectors will be created at CHORI by recombineering of BAG clones and will contain exchangeable modules based on Gateway(tm) technology. The vectors will be transferred into ES cells at Sanger and sequenced in full. Each target locus will be tagged with a lacZ reporter cassette that effectively disrupts the gene to create a null allele. Over 1 million ES cell colonies will be robotically arrayed during the course of the project. Allele structures will be confirmed through long-range PCR for up to 100 ES cell clones per targeting experiment. Five independent knock-out mutants will be archived for each gene to maximize the likelihood of germ-line transmission. Comprehensive quality assurance testing of 300 ES mutant lines annually in vitro and in vivo will take place under the direction of the MBP-UCD to ensure pluripotency, establish germ-line transmission, and confirm viability of live mice and cryopreserved embryos and sperm. Mutant ES cells and embryos will be split and placed in cryo-storage between Sanger and UCD, while modular targeting vectors will be stored at CHORI.

描述：（由申请人提供）我们提出，仅基因靶向创建了10,000个小鼠基因的基因敲除等位基因。目前，从C57BL/6衍生的胚胎干细胞中产生突变小鼠，对于该项目不足以稳健。因此，我们将从源自高效的129小鼠菌株的ES细胞开始，一旦对高通量基因靶向进行验证，便会将其切换到C57BL/6 ES细胞。这项工作将由加利福尼亚州奥克兰的奥克兰研究所的三个成员联盟（CHORI），惠康信托基金会，欣克斯顿，剑桥，英国和加利福尼亚州戴维斯大学老鼠生物学计划（UCD-MBP）进行。定位向量将通过重新组合袋子克隆来创建，并包含基于Gateway（TM）技术的可交换模块。向量将在Sanger处转移到ES细胞中，并完整测序。每个目标基因座都将用LACZ报告基因盒有效地破坏基因以创建无效等位基因。在项目过程中，将超过100万个ES细胞殖民地机器人数量。每个靶向实验将通过远程PCR确认等位基因结构，以获得多达100 ES细胞克隆。每个基因将存档五个独立的敲除突变体，以最大程度地提高种系传输的可能性。每年在体外和体内进行300 ES突变线的综合质量保证测试将在MBP-UCD的指导下进行，以确保多能性，建立生殖线传播并确认活小鼠和冷冻保存胚胎和精子的生存能力。突变的ES细胞和胚胎将被分开并放置在Sanger和UCD之间的冷冻存储中，而模块化靶向矢量将存储在Chori上。