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Tuesday, April 24 • 12:00pm - 1:30pm
Method For Isolation Of Vomeronasal-2 Receptors From Vomeronasal Organ Cdna Library For Eventual Expression In Mammalian Cells

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The vomeronasal organ (VNO) is a chemosensory organ present in amphibians, reptiles, and non-primate mammals. In mice, vomeronasal neurons express vomeronasal-1 receptors (V1R) or vomeronasal-2 receptors (V2R), both of which are G protein-coupled receptors involved in pheromone detection. V2Rs are expressed by the basal neurons of the VNO. They are of special interest because they are used to detect protein pheromones, the Major Urinary Proteins (MUPs), which induce intermale aggression, female responsiveness to mating, and territory marking behaviors. Because V2Rs use combinatorial coding instead of a labelled line coding strategy, linking pheromone responses to the correct V2R has so far been difficult. We designed primers for each V2R to amplify separate individual sequences from a cDNA library, which could then be transfected into cells using vectors. This cell culture method would allow for deorphanization of V2Rs by creating entire cell populations which only express a single V2R. With V2Rs deorphanized, mapping of pathways can begin from a bottom-up method, instead of the more difficult top-down approach. Here we show how to isolate and clone V2Rs for individual eventual expression in mammalian cells using DNA purification, Zero Blunt TOPO cloning PCR kit and ligation into mammalian vector. V2R 83 was successfully cloned into a bacterial vector with a complete sequence. Phosphatase treatments aided correct insert directionality when the V2R was transferred into mammalian vectors using blunt end cloning. Our results demonstrate progress towards setting up a cell culture based V2Rs expression system. This system will allow for further research to deorphanize individual V2Rs by matching them to their corresponding ligands. Stimulation of V2Rs reproducibly activate specific neural circuits in mouse brains, allowing for reliable study of how external environmental cues can direct behaviors. Cloning V2Rs is the first step to identify receptor-ligand interactions, which can be utilized for future research.


Tuesday April 24, 2018 12:00pm - 1:30pm PDT
Sherrill Center Concourse

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