High-throughput targeted mutagenesis of mouse stem cell lines

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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.