The mammalian response to sepsis depends on multiple pathways with genes that contribute to the response. The function of most genes involved in sepsis-induced multiple organ failure are unknown. Mutations provide key information about the function of a gene. Using mutation-based research to determining which genes are involved in livestock's response to sepsis is difficult. Therefore, a comparative approach will be used to identify key livestock genes. Candidate genes will be identified in a model organism amenable to mutagenesis, the mouse. Chromosome engineering and N-ethyl-N-nitrosourea (ENU) mutagenes will be combined to generate new mouse mutants. Since the liver and cells of myeloid lineage are intricately involved in the response to sepsis and multiple organ failure, mutant mice will express only the mutant form of genes in these cell types. This feature allows detection of genes important in response to sepsis that might not be detected if the gene has multiple functions and is essential in development.
NON-TECHNICAL SUMMARY: The spread of bacterial infection to the whole body in livestock can severly impact animal health and can result in death. This is especially important if livestock become targets of bioterrorism attacks. The purpose of this project is to identify genes in livestock important in cntrolling the response to sepsis.
APPROACH: Distal mouse Chromosome 2 has been chosen as the test region in this new mutagenesis approach. Cre-lox and embryonic stem (ES) cell technologies will be combined to target a 56 Mb region for deletion and inversion. Mutations generated by ENU mutagenesis will be uncovered in mice with large deletions occurring exclusively in hepatocytes or myeloid cells. Detection of mutants will involve use of phenotype screens for sensitivity or resistance to sepsis-induced multiple organ failure. Mice with mutant genes that produce variant responses to sepsis will be maintained as heterozygotes with the 56 M inversion to ensure no loss of mutation due to recombination. Mutants will be fine mapped using mice capable of producing smaller hepatocyte or myeloid-specific deletions within the 56 Mb region. All mice capable of producing smaller tissue-specific deletions will be derived from ES cells and will utilize the Cre-lox system. Mutant genes will be isolated and identified using high-throughput techniques and sequencing based on mouse sequence databases. Orthologs will be identified in cattle and pigs and used as candidates for future studies of sepsis-induced multiple organ failure.