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: 7921328
• 负责人: Pieter J. de Jong
• 金额: $ 510万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921328
• 关键词: Administrative Supplement; Alleles; Animal Model; Archives; Breeding; Cell Line; Chimera organism; Communities; Country; Data; Deposition; ES Cell Line; Embryo; Employment; Equipment; Funding; Gene Expression; Gene Expression Profile; Genetic; Goals; Health; Human; Human Resources; Knockout Mice; LacZ Genes; Measures; Microscopic; Modeling; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Organ; Phenotype; Progress Reports; Protocols documentation; Research; Research Personnel; Staging; Staining method; Stains; Stem cells; Supported Employment; Therapeutic; Tissue-Specific Gene Expression; Tissues; Work; embryonic stem cell; human disease; mature animal; mouse model; mutant; novel diagnostics; programs; repository; sperm cell

The KOMP team at UC Davis proposes to convert up to 212 unique ES cell lines over 2 years (106 lines each year) into mutant mouse lines and conduct gene expression analysis, transcriptome analysis, breeding to homozygosity, and creation of a cryopreserved archive of germplasm. First, we shall microinject ES cell clones to derive chimeras for generating germline-confirmed, heterozygous knockout mouse lines; second, we shall perform whole mount LacZ expression on late-stage embryos and/or stain for LacZ on microscopic sections of specific organs from adult animals in order to phenotype tissue-specific gene expression of the targeted allele; third, heterozygous mice will be intercrossed to genetic homozygous mutants of the targeted allele in order to distinguish between embryonic lethal and developmentally competent mutations, the latter which will be used to create cryopreserved embryos and sperm for archival deposition and distribution to the research community; and fourth, we shall perform transcriptome analysis on 106 select LacZ-expressing tissues from homozygous mutant mice. We agree to quarterly reporting of progress as stipulated in the ARRA program generally described at http://www.recovery.gov/. Hypothesis-driven phenotyping will be done by subject experts, namely research investigators across the country who order the mice. Thus, this supplement is focused on accelerating activities within the original scope of work of the KOMP mutagensis program with specific goals and targets, and will not pay for less specific or more specialized phenotyping activities (that often requires specialized equipment or expertise, possibly specific environmental challenges for the phenotype to appear) or for the creation of alternative phenotyping protocols. This project will also support the employment of more than 5 new technical staff and academic personnel over 2 years. The products (e.g., mice) generated by this project will be available through the KOMP repository for purchase, thereby generating funds that can be applied to continued employment of new hires.

加州大学戴维斯分校的KOMP团队建议转换多达212个唯一的ES细胞 将2年以上的品系(每年106个品系)转化为突变小鼠品系并传导基因 表达分析、转录组分析、纯合育种和创造 一个超低温保存的种质档案。首先，我们将显微注射ES细胞克隆到 获得用于产生生殖系确认的杂合基因敲除小鼠品系的嵌合体； 第二，我们将在晚期胚胎和/或 在成年动物特定器官的显微镜切片上进行LacZ染色 目标等位基因的表型组织特异性基因表达；第三，杂合子小鼠 会与目标等位基因的遗传纯合突变体杂交，以便 区分胚胎致命性突变和发育能力突变， 后者将用于创建冷冻保存的胚胎和精子以供存档 沉积和分发给研究界；第四，我们将执行 106例纯合突变型LacZ表达组织的转录组分析 老鼠。我们同意按照ARRA计划的规定每季度报告进展情况 一般在http://www.recovery.gov/.上描述假说驱动的表型将是 由主题专家完成，即全国各地的研究调查人员下令 老鼠。因此，本补编侧重于加速原文中的活动。 KOMP突变方案的工作范围，具有具体的目标和指标，并将 不支付不太具体或更专业的表型鉴定活动(这通常需要 专门的设备或专业知识，可能是特定的环境挑战 将出现的表型)或创建替代表型方案。这个项目 还将支持雇用5名以上新的技术人员和学术人员 2年以上人事管理经验。该项目产生的产品(例如MICE)将是 可通过KOMP储存库购买，从而产生资金，可以 适用于新员工的续聘。