Abstract

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 differ from V1Rs in their sequence length and G-protein linkage, and are of special interest because they are used to detect proteins, major urinary proteins (MUPs), which induce intermale aggression, female responsiveness to mating, and territory marking. Because V2Rs use combinatorial coding instead of a labelled line, linking pheromone responses to the correct V2R has so far been difficult; even though each cell expresses a different V2R, each V2R could respond to multiple MUPs and each MUP could activate multiple V2Rs. This paper describes a cell culture method that would allow for deorphanizing 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. This paper shows how to isolate and clone V2Rs for eventual individual expression in mammalian cells using DNA purification, Zero Blunt TOPO cloning and ligation into mammalian vector. V2Rs 83 and 121 were cloned into a bacterial vector. Currently, work is being done to understand if the blunt end cloning did result in capturing of a complete V2R sequence in the correct orientation for future transfer to mammalian vectors. These results demonstrate progress towards setting up a cell culture based V2Rs expression system. This system will allow for further research on the identification of receptor-ligand interactions, enabling a reliable study of how external environmental cues can direct behaviors.

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