High-throughput targeted mutagenesis of mouse stem cell lines

我们建议专门通过基因打靶来创建10,000个小鼠基因的敲除等位基因。这个 从C57BL/6来源的胚胎干细胞产生突变小鼠目前还不够健壮 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 一旦C57BL/6 ES细胞被验证可以进行高通量的基因打靶，就可以立即进行基因打靶。这项工作将是 由加利福尼亚州奥克兰的奥克兰儿童医院研究所的三名成员组成的财团完成 (CHORI)、威康信托桑格研究所、英国剑桥欣克斯顿和加州大学戴维斯分校 小鼠生物学计划(UCD-MBP)。靶向载体将通过重组BAG在CHORI创建 克隆并将包含基于Gateway？技术的可交换模块。向量将被转移 进入桑格的胚胎干细胞，并进行了全序列测定。每个目标轨迹都将被标记一个LacZ报告卡带， 有效地破坏基因以产生零等位基因。100多万个ES细胞克隆将被机器人排列 在这个项目的过程中。将通过对多达100个ES细胞进行远程聚合酶链式反应来确认等位基因结构 每个目标实验的克隆数。将为每个基因存档五个独立的敲除突变体 最大限度地提高生殖系传播的可能性。300株ES突变体的全面质量保证检测 每年在体外和体内的品系将在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胚胎和精子的存活率。 突变的胚胎干细胞和胚胎将被分裂并放置在桑格和UCD之间的冷冻库中，同时模块化 靶向载体将存储在CHORI。

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.