High-throughput targeted mutagenesis of mouse stem cell lines

描述：(申请者提供)我们建议通过基因打靶的方法创造10,000个小鼠基因的敲除等位基因。从C57BL/6来源的胚胎干细胞产生突变小鼠目前对该项目来说还不够强大。因此，我们将从来自高效129小鼠品系的ES细胞开始，一旦C57BL/6 ES细胞被验证可以进行高通量基因打靶，就切换到C57BL/6 ES细胞。这项工作将由加州奥克兰的儿童医院奥克兰研究所(CHORI)、英国剑桥欣克斯顿的威康信托桑格研究所(Wellcome Trust Sanger Institute)和加州大学戴维斯分校(UCD-MBP)的三名成员组成的财团完成。目标载体将通过重组BAG克隆在CHORI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ES细胞中并进行全序列测定。每个靶基因将被标记一个LacZ报告盒，该盒有效地扰乱基因，产生零等位基因。在项目过程中，100多万个胚胎干细胞克隆将被机器人排列。每个靶向实验将通过长达100个ES细胞克隆的远程聚合酶链式反应来确认等位基因结构。将为每个基因存档五个独立的敲除突变，以最大限度地增加生殖系传播的可能性。在MBP-UCD的指导下，每年将在MBP-UCD的指导下对300个ES突变系进行体内外全面的质量保证测试，以确保多能性，建立生殖系传播，并确认活鼠和冷冻胚胎和精子的存活能力。突变的ES细胞和胚胎将被分裂并放置在桑格和UCD之间的冷冻库中，而模块化靶向载体将被存储在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.