Isolation andIn VitroDifferentiation of Conditionally Immortalized Murine Olfactory Receptor Neurons

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Abstract
Two major challenges exist in our understanding of the olfactory system. One concerns the enormous combinatorial code underlying odorant discrimination by odorant receptors. The other relates to neurogenesis and neuronal development in the olfactory epithelium. To address these issues, continuous cell cultures containing olfactory receptor neurons (ORNs) were obtained from olfactory epithelia of H-2Kb-tsA58 transgenic mice. ORNs were detected and characterized by immunocytochemistry, RT-PCR, and Western blot for the markers Gαolf, adenylyl cyclase III, the olfactory cyclic nucleotide-gated channel subunits, and olfactory marker protein. In culture, epidermal growth factor and nerve growth factor stimulated proliferation, and brain-derived neurotrophic factor and neurotrophin-3 induced cellular maturation.Clonal cell lines were isolated by fluorescence-activated cell sorting with anti-neural cell adhesion molecule antibodies, and of 144 single cells plated, 39 clones were expanded, propagated, and stored in liquid nitrogen. All attempts at recovery of clonal lines from frozen stocks have been successful. The most thoroughly characterized clone, 3NA12, expressed ORN markers and responded to stimulation by single odorants. Each odorant activated ∼1% of cells in a clonal line, and this suggests that many different odorant receptors may be expressed by these clonal cells. Therefore, these cell lines and the method by which they have been obtained represent a significant advance in the generation of olfactory cell cultures and provide a system to investigate odorant coding and olfactory neurogenesis.