High-throughput targeted mutagenesis of mouse stem cell lines

描述：（由申请人提供）我们提出仅通过基因靶向产生小鼠基因的10，000个敲除等位基因。从C57 BL/6衍生的胚胎干细胞产生突变小鼠目前对于该项目来说还不够稳健。因此，我们将从来自高效129小鼠品系的ES细胞开始，一旦它们被验证用于高通量基因靶向，就切换到C57 BL/6 ES细胞。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（CHORI）、英国剑桥Hinxton的Wellcome Trust桑格研究所和加州大学戴维斯分校小鼠生物学项目（UCD-MBP）的三名成员组成的财团完成。靶向载体将在CHORI通过BAG克隆的重组工程来创建，并且将包含基于Gateway（tm）技术的可交换模块。载体将在桑格转移到ES细胞中并进行完全测序。每个靶基因座将用lacZ报告盒标记，其有效地破坏基因以产生无效等位基因。超过100万个ES细胞集落将在项目过程中自动排列。等位基因结构将通过长距离PCR确认，每次靶向实验最多100个ES细胞克隆。每个基因将存档5个独立的敲除突变体，以最大限度地提高生殖系传播的可能性。在MBP-UCD的指导下，每年将在体外和体内对300个ES突变株系进行全面的质量保证测试，以确保多能性，建立种系传播，并确认活小鼠和冷冻保存的胚胎和精子的活力。突变ES细胞和胚胎将被分离并置于桑格和UCD之间的冷冻储存中，而模块化靶向载体将储存在CHORI。

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.