Development of a High Throughput Insect Olfactory Receptor Deorphanization Method through Ectopic Expression in Drosophila melanogaster’s Olfactory Sensory Neurons

Diplôme Master Development of a High Throughput Insect Olfactory Receptor Deorphanization Method through Ectopic Expression in Drosophila melanogaster’s Olfactory Sensory Neurons eng 2023-08-24 2023-08-09 author Faoro Nicolas Mathieu 899675 director Rodriguez Ivan 28183 Département de génétique et évolution (Genev) (BIANI) 49 Insect olfaction is today only partially understood. In particular, a very limited number of odorant receptor-agonist pairs have been determined. Typically, the investigation of odorant receptors (ORs) involves electrophysiology techniques using model organisms like Drosophila melanogaster. However, electrophysiology has inherent limitations, including its complexity, time-consuming nature, and the requirement to test individual antennas or sensilla. These constraints pose significant challenges for deorphanizing large OR repertoires and analyzing combinatorial codes, which necessitate testing multiple odorants against many receptors. To address these challenges, we present an alternative to electrophysiology, offering a rapid and efficient method for analyzing complete receptor repertoires against diverse odorant panels. In our approach, we expressed exogenous insect ORs in the antennas of Drosophila melanogaster and combined it with calcium imaging using optimized transgenic lineage production. To streamline the analysis process, we developed a single transgene containing both a reporter and an odorant receptor. Additionally, we introduced an empty neuron system to mitigate endogenous noise from the Drosophila repertoire. Our results demonstrate comparable sensitivity and specificity to electrophysiology, providing validation for the effectiveness of our methodology. Moreover, we achieved expedited fly generation and high-throughput analysis by simultaneously testing multiple antennas, each harboring different genotypes, against a sequence of odorants controlled by an olfactometer. While the empty neuron system requires further optimization due to low signals, it effectively reduced endogenous noise. In conclusion, our methodology offers a rapid and efficient approach for analyzing complete odorant receptor repertoires in insects. It enables the expedited deorphanization of numerous OR-odorant couples, requiring fewer manipulations and shorter analysis times compared to electrophysiology techniques. Our approach holds promise for advancing the field of insect olfaction, facilitating the analysis of repertoires in diverse winged insect species. While further optimization of the empty neuron system is warranted, our methodology presents a valuable tool for accelerating research in the fascinating realm of insect olfaction. Insect Olfaction Polycistronic Transgene Calcium Imaging Empty Neurons Deorphanization Combinatorial Code Odorant Receptors Dewey 590 Master en Biologie Option Génétique, Développement, Evolution 44 Département de génétique et évolution 162 false 820 Groupe Rodriguez f26cb560-6109-4eca-b68b-b929d05dbe58 Master thesis Private 09_08_23_FAORO_Nicolas_Master_Thesi.pdf 520961 application/pdf c8b6c614ff58218239a7264520338e4615cb48d6b5820c409192aa4a78f8a6a7 closedAccess Faculté des sciences / Section de biologie / Département de génétique et évolution

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Development of a High Throughput Insect Olfactory Receptor Deorphanization Method through Ectopic Expression in Drosophila melanogaster’s Olfactory Sensory Neurons				2023	64	0

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