CHILDREN'S HOSPITAL & RESEARCH CENTER

The purpose of this project is to extend the scientific value of the resources generated by the Knockout Mouse Project (KOMP). The overall aim of KOMP is to dramatically increase the number of unique knockout alleles crucial to many fields of translational research by creating between 5,000-8,500 new and unique gene targeted knockout ES cell lines over the next 5 years. Prioritization of genes for targeting in the KOMP program was achieved by polling the scientific community in a variety of fields of study (e.g., neurology, infectious disease, immunology, metabolism, etc). Thus, the genes to be knocked out have already been implicated in one or more human diseases, but their specific role in the complex pathways and networks of cells and live mammals need validation in the mouse model before therapeutic and diagnostic applications are developed for human patients. Since most researchers study knockout alleles in live mice, the scientific value of this resource could be greatly enhanced by accelerating the conversion of mutant ES cells into knockout mice. Once generated, gene expression analysis and determination of embryonic lethality can be conducted at relatively minimal cost on live mice prior to preservation as frozen germplasm for deposition in the distribution archive. Therefore, this project will generate 212 unique mutant mouse lines from KOMP program-generated ES cells for gene expression analysis, cryopreservation, and distribution. First, we shall convert ES cell lines into genotype-confirmed, heterozygous knockout mouse lines; Second, we shall perform whole mount LacZ expression on late-stage embryos and on microscopic sections of organs from adults in order to analyze tissue-specific gene expression of the targeted allele; Third, heterozygous mice will be intercrossed to genetic homozygosis at the targeted allele in order to distinguish between mutations that are embryonic lethal and those that are developmentally competent, the latter which will be used to create cryopreserved embryos and sperm for archival deposition and distribution to the research community. In 100 homozygous mutants, transcriptome analysis will be completed in a subset of three or more adult tissues that express LacZ. 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 repository with specific goals and targets. Data and information generated from analysis of gene expression and embryonic lethality will add substantive scientific value to the KOMP resource. Further, this project will make genotypically-confirmed, germline transmitted, specific pathogen-free knockout mice readily available for distribution, will establish baseline gene-expression and developmental data and information in an easily accessible and searchable manner online, and will produce a distribution archive of germplasm to preserve and protect the mutant mouse lines. In addition, this project will derive live mice faster, more economically, and with a higher likelihood of success at a specialized and centralized facility with established expertise than could be done by any one individual. This approach also will save researchers significant time and money by confirming germline transmission, creating live mice for study, and generating useful baseline gene expression and developmental data.

Clarification of Codes