High-throughput targeted mutagenesis of mouse stem cell lines

描述：（由申请人提供）我们建议通过基因靶向专门创建10,000个小鼠基因的敲除等位基因。从C57BL/6衍生的胚胎干细胞中产生突变小鼠目前还不足以用于该项目。因此，我们将从高效的129小鼠品系衍生的胚胎干细胞开始，一旦它们被验证具有高通量基因靶向性，就切换到C57BL/6胚胎干细胞。这项工作将由加州奥克兰儿童医院奥克兰研究所（CHORI）、英国剑桥Hinxton的Wellcome Trust Sanger研究所和加州大学戴维斯分校小鼠生物学计划（UCD-MBP）的三名成员组成的联盟完成。靶向载体将在CHORI通过重组BAG克隆来创建，并将包含基于Gateway（tm）技术的可交换模块。这些载体将被转移到桑格的胚胎干细胞中并进行完整测序。每个目标位点将被标记一个lacZ报告磁带，有效地破坏基因，创造一个空等位基因。在项目过程中，将有超过100万个胚胎干细胞菌落被机器人排列。每个靶向实验将通过多达100个胚胎干细胞克隆的远程PCR来确认等位基因结构。每个基因的5个独立的基因突变将被存档，以最大限度地提高种系传播的可能性。在MBP-UCD的指导下，每年对300个ES突变系进行体外和体内的全面质量保证测试，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。突变的胚胎干细胞和胚胎将被分裂并放置在Sanger和UCD之间的冷冻储存中，而模块化靶向载体将存储在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.