High-throughput targeted mutagenesis of mouse stem cell lines

加州大学戴维斯分校的KOMP团队建议将多达212个独特的ES细胞 品系（每年106个品系）转化为突变小鼠品系和传导基因 表达分析、转录组分析、纯合性育种和创造 冷冻保存的种质档案。首先，我们将显微注射ES细胞克隆， 衍生嵌合体以产生种系确认的杂合敲除小鼠系; 第二，我们将在晚期胚胎上进行整批LacZ表达，和/或 对成年动物特定器官的显微切片进行LacZ染色， 靶向等位基因的表型组织特异性基因表达;第三，杂合子小鼠 将与靶向等位基因的遗传纯合突变体杂交， 区分胚胎致死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 向研究界提供沉积和分配;第四，我们将执行 106例LacZ纯合突变体组织转录组分析 小鼠我们同意按照ARRA计划的规定，每季度报告进展情况 通常在http://www.recovery.gov/中描述。假设驱动的表型分析将是 由主题专家完成，即全国各地的研究人员， 小鼠因此，这一补充的重点是加快活动内的原始 KOMP诱变项目的工作范围，具有特定的目标和指标，并将 不支付不太具体或更专门的表型分析活动（这往往需要 专业设备或专门知识，可能是特定的环境挑战， 表型出现）或用于创建替代表型分析方案。这个项目 还将支持雇用5名以上的新技术人员和学术人员 人员2年以上。产品（例如，该项目产生的小鼠）将 可通过KOMP存储库购买，从而产生资金， 适用于继续雇用新员工。

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.