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: 7455583
• 负责人: Pieter J. de Jong
• 金额: $ 2.88万
• 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/7455583
• 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; Numbers; Operative Surgical Procedures; Pediatric Hospitals; Performance; Phenotype; Polymerase Chain Reaction; Process; Production; Public Domains; Purpose; Quality Control; Range; Rate; 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; member; mouse genome; mutant; null mutation; parallel processing; pathogen; pluripotency; programs; quality assurance; research study; size; sperm cell; technological innovation; transmission process; vector

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¿ 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.

我们建议专门通过基因靶向创建10，000个小鼠基因敲除等位基因。的 从C57 BL/6衍生的胚胎干细胞产生突变小鼠目前对此还不够稳健 项目因此，我们将从来自高效129小鼠品系的ES细胞开始，切换到 C57 BL/6 ES细胞，一旦它们被验证用于高通量基因靶向。这项工作将 由位于加利福尼亚州奥克兰的儿童医院奥克兰研究所的三人联合会完成 （CHORI）、英国剑桥欣克斯顿威康信托桑格研究所和加州戴维斯大学 小鼠生物学程序（UCD-MBP）。靶向载体将在CHORI通过BAG的重组工程创建 克隆，并将包含基于网关技术的可交换模块。带菌者会被转移到 在桑格的胚胎干细胞中进行全序列测定。每个靶基因座将用lacZ报告盒标记， 有效地破坏基因以产生无效等位基因。超过100万个胚胎干细胞集落将被机器人排列 在项目的过程中。等位基因结构将通过最多100个ES细胞的远程PCR进行确认 克隆/靶向实验。每个基因将存档五个独立的敲除突变体， 最大化生殖系传播的可能性。300个ES突变体的全面质量保证测试 每年在体外和体内的细胞系将在MBP-UCD的指导下进行，以确保多能性， 建立生殖系传播，并确认活小鼠和冷冻保存的胚胎和精子的活力。 突变的ES细胞和胚胎将被分裂并放置在桑格和UCD之间的冷冻储存中，而模块化 靶向载体将储存在CHORI。