Genetic and proteomic analysis of inflammation

Vanderbilt University, Nashville TN

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Abstract

Genome-wide genetic and proteomic studies are now possible as a result of efforts to sequence the human and mouse genomes. Both approaches will be used to characterize genes and functional interactions among proteins that are important in inflammation. To assess gene function, a process of tagged-sequence mutagenesis has been developed. Libraries of embryo-derive stem (ES) cell clones will be isolated in which cellular genes have been disrupted by a gene trap retrovirus vector. The resulting libraries of sequenced mutations offer the immediate potential of constructing mice with germline mutations in specific genes of interest. Initial experiments will use an existing library which contains over 600 characterized mutations, including more than 120 that involve anonymous ESTs and 24 that involve known genes of potential importance to blood cell biology. Subsequent experiments will employ vectors designed to target most genes regardless of whether they are expressed in ES cells. cDNAs corresponding to each disrupted gene will be applied to high density DNA microarrays to identify genes in the mutant library whose expression is restricted to blood cells, is altered during blood cell activation and inflammation, or is altered in mice that harbor mutations in specific genes. Approximately 15 mutations in ES cells will be characterized per year, of which 15 will be selected for transmission into the mouse germline. The resulting knockout mice will be screened for phenotypes affecting blood cell development and responses to inflammatory stimuli. Proteins encoded by genes important in blood cell biology and inflammation will be analyzed to identify cellular proteins with which they interact. First, genes disrupted by tagged sequencer mutagenesis will be engineered by replacing the inserted retrovirus with sequences that will drive the expression the occupied gene as an affinity tagged fusion protein. Alternatively, purified affinity tagged proteins will be introduced directly into cells by protein transduction. Intracellular complexes containing the affinity tagged proteins will then be purified and the interacting proteins will be identified by mass spectrometry. The functional analysis of inflammatory genes at both biological and biochemical levels will improve our understanding of the disease process at a molecular level, and suggest targets for therapeutic intervention.