Odorant receptor (OR) genes form the largest family in the mouse genome: ~1200 genes spread over ~40 loci. Each olfactory sensory neuron (OSN) expresses one OR gene, from one allele. The mechanisms of OR gene choice remain elusive. We will execute five specific aims that are interconnected but independent. We will search for homeodomain genes that we can link functionally to expression of a subset of OR genes; we will define promoter regions for the eight OR genes that are solitary, not belonging to a cluster; we will look for organizational principles among the repertoire of second choices in OSNs that express first an OR locus without a coding sequence; we will characterize the phenotype of mice with a knockout of a novel regulatory element, the P element; and we will test the distance-dependence of the activity of this and a similar element (the H region) by transplanting it within the local genomic region. Guiding hypotheses are that promoter regions for OR genes are short and close to the coding sequence; that the conserved homeodomain and O/E binding sites in OR promoter regions have a fundamental role in OR gene choice, rather than in transcription after it is chosen for expression; and that the H and P elements are two of several similar regulatory elements that each operate in cis within a cluster. The approach is based on gene targeting and transgenesis by pronuclear injection. A multipronged strategy will be taken to assay OR gene expression, with βgal-reporter mice, in situ hybridization, custom Affymetrix microarrays for mouse ORs, quantitative, real-time PCR, and Nanostring molecular bar codes. Understanding OR gene choice will have implications for our understanding of the regulation of gene expression in the mammalian genome – particularly if new mechanisms or principles are discovered.
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