William C. Skarnes - US grants

laboratory mouse; tissue resource /registry

The NHLBI-Bay Area Functional Genomics Consortium will use gene-trap vectors to inactivate thousands of genes in mouse embryonic stem (ES) cells and make them freely available for the purpose of generating knockout mice. In preliminary studies, custom gene-trap vectors have been used to trap more than 500 mouse genes, some completely novel, and many corresponding to ESTs of unknown function. Approximately 150 of the "trapped" ES cell clones have been transmitted through the germline, and studies of the knockout mice have already led to the identification of completely novel genes that are important in cardiopulmonary development and disease. The Consortium involves several leading San Francisco Bay Area research institutions: The J. David Gladstone Institutes, the University of California, San Francisco, and the University of California, Berkeley. The Consortium is organized into nine Components: (1) Gene Trapping in Embryonic Stem Cells, (2) Computational Methods for Predicting Gene Function, (3) In Situ Hybridization, (4) Gene Expression Profiling and Analysis, (5) Mouse Resource for Pulmonary Disease, (6) Mouse Resource for Lipid Metabolism and Atherogenesis, (7) Mouse Resource for Cardiopulmonary Development, (8) Cardiopulmonary Genomics Education, and (9) Administration. The major objective of the Consortium (corresponding to Component 1) is to use custom gene-trap vectors to inactivate at least 2,500 genes per year in ES cells. Each "trapped" ES cell line will be posted on the Consortium's website (genetrap.org) and will be distributed freely to the research community for the purpose of producing knockout mice. A second objective (corresponding to Components 2-4) is to assess which of the ES cell lines is likely to be valuable for understanding cardiopulmonary development and common cardiopulmonary diseases. To achieve this objective, the investigators will use computational approaches, expression profiling with DNA microarrays, and in situ hybridization studies. A third objective (corresponding to Components 5-7) is to select a few ES cell clones for the production of knockout mice, for the purpose of understanding genes involved in cardiopulmonary development and disease. The Consortium's resources will be distributed freely to any interested investigator and should provide a catalyst for many different NHLBI-funded research programs.

DESCRIPTION (provided by applicant): Gene trapping in mouse embryonic stem cells offers a powerful tool to create insertional mutations in mice that are immediately accessible to molecular analysis. However, certain deficiencies in the technology have been recognized and need to be addressed before embarking on a large-scale effort to trap each of the 30,000 or so genes in the mammalian genome. These drawbacks include the incompatibility of the mouse strains used for gene trapping (and gene targeting) in relation to classical ENU mutagenesis efforts now underway. More importantly, the versatility of conventional gene trapping approaches is limited and does not allow for the generation of other, more useful, alleles that are needed to fully address gene function. The goal of this grant is to develop optimal gene trap vector designs and methods for the large-scale functional analysis of genes in mice. Specifically, this proposal aims to isolate highly germline-competent, feeder-independent embryonic stem cells from the C57BL/6 strain of mice, to construct "universal" gene trap vectors capable of targeting all classes of genes, and to design and test the feasibility of gene trap vectors with site-specific recombination sites. The strategic placement of site-specific recombination sites will permit secondary modifications at the gene trap locus enabling the creation an unlimited variety of desirable alleles. These modifications include, but are not limited to, 1) the generation of an allelic series of mutations in the trapped gene (null, hypomorphic, and site-specific mutations), 2) the creation of conditional alleles (temporal and tissue-specific), 3) the inclusion of reporter genes for the study of gene expression and cellular functions (Bgal, GFP, PLAP, etc.), 4) the introduction and controlled expression of recombinases (Cre, fip, etc.) and transactivators (TetR, gal4, etc.), 5) the production of knock-ins of heterologous gene products, and 6) the addition of negative drug selection cassettes (HPRT, TK) for engineering chromosomal deletions and inversions. The technology described in this proposal will have wide applications to all areas of mammalian biology and will complement and extend other mutagenesis strategies in mice.

The NHLBI-Bay Area Functional Genomics Consortium will use gene-trap vectors to inactivate thousands of genes in mouse embryonic stem (ES) cells and make them freely available for the purpose of generating knockout mice. In preliminary studies, custom gene-trap vectors have been used to trap more than 500 mouse genes, some completely novel, and many corresponding to ESTs of unknown function. Approximately 150 of the "trapped" ES cell clones have been transmitted through the germline, and studies of the knockout mice have already led to the identification of completely novel genes that are important in cardiopulmonary development and disease. The Consortium involves several leading San Francisco Bay Area research institutions: The J. David Gladstone Institutes, the University of California, San Francisco, and the University of California, Berkeley. The Consortium is organized into nine Components: (1) Gene Trapping in Embryonic Stem Cells, (2) Computational Methods for Predicting Gene Function, (3) In Situ Hybridization, (4) Gene Expression Profiling and Analysis, (5) Mouse Resource for Pulmonary Disease, (6) Mouse Resource for Lipid Metabolism and Atherogenesis, (7) Mouse Resource for Cardiopulmonary Development, (8) Cardiopulmonary Genomics Education, and (9) Administration. The major objective of the Consortium (corresponding to Component 1) is to use custom gene-trap vectors to inactivate at least 2,500 genes per year in ES cells. Each "trapped" ES cell line will be posted on the Consortium's website (genetrap.org) and will be distributed freely to the research community for the purpose of producing knockout mice. A second objective (corresponding to Components 2-4) is to assess which of the ES cell lines is likely to be valuable for understanding cardiopulmonary development and common cardiopulmonary diseases. To achieve this objective, the investigators will use computational approaches, expression profiling with DNA microarrays, and in situ hybridization studies. A third objective (corresponding to Components 5-7) is to select a few ES cell clones for the production of knockout mice, for the purpose of understanding genes involved in cardiopulmonary development and disease. The Consortium's resources will be distributed freely to any interested investigator and should provide a catalyst for many different NHLBI-funded research programs.

DESCRIPTION (provided by applicant): The K0MP2 data are a critical resource for biomedical research, will inform human disease studies, be integrated with existing resources and will be accessed, analyzed and mined by mouse biologists, translational researchers, clinicians and wider biomedical community. The MIP2 project will support the K0MP2 project by providing the Data Coordination Centre which will process the complex phenotypic data provide access via a web portal. The DCC aims are to collect and store valid data as it appears throughout the project, to provide unified access to these data for specialist and non specialist users via the web and programmatically, and to support complex queries and statistical analyses. The project has several distinct components and tasks: The Pheno-DCC will validate, perform quality control and manage data acquired dynamically from centres. This will ensure data are robust and allow progress tracking of data at all stages of processing. Specialist data wranglers will manage this process and will interact with the users of the data to ensure user interfaces support the needs of the varied community who will access these data. The statistical and annotation pipelines and environment which will analyze raw data and summarize data for each mutant and assay for presentation to users. A supporting core database, the Core Data Archive, which will store all project data, provide programmatic access, push data to external resources such as NCBI and the Jackson Laboratory and critically provide data for the user interfaces. A single point of entry web portal hosted at www.knockoutmouse.org which will present data to users. I, integrate data from parallel mouse phenotyping projects and provide access to a statistical analysis and query environment Integrate K0MP2 data via biomedical databases at the EBI to ensure that the data are widely distributed to mouse biologists and other scientists .