Effect of reducing insulin signaling on third instar larval OSN gene expression in Drosophila melanogaster
Summary : We characterized insulin receptor (InR)-dependent gene expression in the Drosophila olfactory sensory neurons (OSNs) using transgenic RNAi.Chronic knockdown of InR in the OSNs was elicited via (Orco-GAL4, UAS-InR-RNAi), and RNA-seq was used to identify potential target genes. ==================================================================================================== Overall design: Drosophila were reared at 25 C and 60% humidity on standard cornmeal-dextrose agar food (Genesee Scientific, #66-112) until the wandering third instar stage. Control (Orco-GAL4; gift from Dr. John Carlson) offspring were compared with (Orco-GAL4 x UAS-InR-RNAi). A UAS-RNAi line targeting InR (VDRC#992) was used. OSNs were isolated using a method described in Slankster et al., 2020. Briefly, we used the UAS/GAL4 expression system to tag OSN nuclei with a KASH-GFP construct; the KASH signal localizes GFP protein expression to the nuclear membrane.Using an affinity-based pull-down approach, the GFP-tagged nuclei were separated from non-OSN nuclei.The enriched OSN nuclei were used as substrates for mRNA extraction. mRNA was extracted to determine the effects of reduced insulin signaling on gene expression using Illumina RNA-seq. ================================================================================================== Contributor(s) Mathew D, Jain R.