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Tuesday, April 24 • 11:20am - 11:40am
Cloning Vomeronasal Type-2 Receptors For Expression And Analysis In A Cell Culture Model System

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The vomeronasal organ (VNO) is an olfactory sense organ in the nose of mice that detects pheromone signals through ligand binding to G-protein coupled receptors. There are three families of VNO receptors, V1R, V2R, and FPR. V2Rs in mice primarily serve to bind large molecules like the major urinary proteins (MUPs), proteins secreted in urine that trigger contextual behaviors in the recipient. Through combinatorial coding, multiple combinations of MUPs can activate multiple V2Rs in different ways, leading to complex signals based on a small library of ligands. However, VNO receptors are orphaned, it is not known which MUP ligand binds with which VNO receptor. This research set out to deorphanize V2Rs and pair them with their cognate ligands to create a library of receptor-ligand pairings. Receptor deorphanization will involve cloning V2Rs into mammalian cells, then analyzing them using patch clamp to measure membrane voltage changes when exposed to MUP ligands. Sequences coding for V2Rs were amplified through PCR, visualized on a gel, relevant bands were extracted and purified, then TOPO cloned into bacterial plasmids and transformed into JM109 E. coli cells for mass growth. Plasmids from E. coli were restriction digested to verify insert sequence length, then ligated into mammalian pEGFP vectors for eventual transfection into eukaryotic cells. To date, receptors have been cloned, visualized, extracted, purified, and transformed for V2Rs 34, 60, 92, 121, 122, 81, and 83. One sample (122-1) indicated a full length sequence, and has been ligated and sent for sequencing. If the results indicate a full length sequence inside the mammalian vector, the plasmid will be transfected for surface expression. This experiment is an important first step to being able to better understand and map the exact neural pathways activated by an environmental chemical stimulus, and how it produces a response in the host.


Tuesday April 24, 2018 11:20am - 11:40am PDT
123 Zeis Hall

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