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科普团队提议转换多达212个独特的ES牢房 在2年以上（每年106条线）中的线条进入突变小鼠线并进行基因 表达分析，转录组分析，繁殖至纯合性和创造 冷冻保存的种质档案。首先，我们将对细胞克隆进行微注射ES克隆 推导用于生成种系确认的杂合敲除小鼠系的嵌合体； 其次，我们将在后期胚胎和/或 lacz在成年动物特定器官的显微镜切片上的染色，以便 靶向等位基因的表型组织特异性基因表达；第三，杂合小鼠 将在目标等位基因的基因纯合突变体中互划，以便 区分胚胎致死和发育能干的突变， 后者将用于创建冷冻保存的胚胎和精子作为档案 向研究界的沉积和分发；第四，我们将表演 对106个从纯合突变体中精选表达LACZ的组织的转录组分析 老鼠。我们同意按照ARRA计划中规定的季度报告进度报告 通常在http://www.recovery.gov/上进行描述。假设驱动的表型将是 由科目专家完成，即全国各地的研究调查员 老鼠。因此，该补充的重点是在原始中加速活动 具有特定目标和目标的Komp诱变计划的工作范围，并将 不支付更少的特定或更专业的表型活动（通常需要 专业设备或专业知识，可能是特定的环境挑战 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位以上的新技术人员和学术 人员超过2年。该项目生成的产品（例如，老鼠）将是 可通过KOMP存储库购买，从而产生可以产生的资金 可用于继续雇用新员工。