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Analysis of the phenotypic effect of SNPs mapping at Irm1 locus which controls IL-1beta production in the mouse.


Mouse lines phenotypically selected for about 70 generations on the basis of High (AIRmax) or Low (AIRmin) acute inflammatory response (AIR), have been useful for studying the genetic control of this innate immune reaction. Linkage analysis using single nucleotide polymorphism (SNP) markers with inflammatory phenotypes, such as quantitative IL-1beta synthesis after inflammasome activation, were carried out in large heterogeneous intercrossed F2(AIRmax x AIRmin) populations. Highly significant linkage signal (LOD score=72) between SNPs and phenotypes mapped a region of about 3.5 Mb at distal chromossome 7, which we nominated Irm1 locus. After next generation sequencing of this genomic region in groups of mice showing extreme high or low response phenotypes, the interval was limited to 450 Kb harboring 14 SNPs. One of these variants maps at an exon of the Pycard gene, whose product is a central element of inflammasome complexes involved in IL-1beta synthesis; others map at intronic regions of integrins and of signaling regulators of G proteins, and others in non-genic regions. The high statistical significance obtained in linkage analysis, together with the fine mapping precision of the Irm1 locus and the reduced numbers of identified SNPs, justify the search for the functional causality of these genetic variants on inflammatory phenotypes that differentiate AIRmax and AIRmin mice. In the present project we intend to make use of CRISPR/Cas9 technology for gene editing, in order to interrogate the function of these polymorphisms on the levels of IL-1beta production, using in vitro assays with murine macrophages that secrete the cytokine after inflammasome complex activation. The technique allows the generation of alleles with precise mutations for functional assays, aiming the establishment of causal links between genetic variants and biological phenotypes. The method has been widely used to evaluate the effect of point mutations on gene function, and thus is perfectly fitted to our main objective in this project. (AU)

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