Masters Thesis

An odor responsive GFP-based reporter for endogenous small interfering RNA activity in the germline

Caenorhabditis elegans forages for food by distinguishing between various odorants in a dynamic environment. Their sensory neurons have the ability to adapt to persistent attractive odors in the absence of food (Colbert and Bargmann, 1995). Adaptation to the odor butanone takes place in the AWC, a paired olfactory sensory neuron (L’Etoile et al., 2002). Olfactory adaptation is initiated by the translocation of a cGMP-dependent protein kinase, EGL-4, from the cytoplasm into the nucleus of the AWC (L’Etoile et al., 2002; Lee et al., 2010). This translocation depends on a decrease in cGMP (O’Halloran et al., 2012). The activity of ODR-1, a transmembrane guanylyl cyclase, which is required for chemotaxis towards all AWC mediated odorants, is down regulated for adaptation (L’Etoile and Bargmann, 2000). Nuclear EGL-4 promotes a 22G RNA directed repression of the odr-1 gene thereby initiating long-term odor adaptation (Juang et al., 2013). mut-7 activity, a 3’-5’ exonuclease, is also implicated in odr-1 22G RNA generation. In addition, a ChIP analysis of HPL-2 was shown to load heterochromatin on the odr-1 locus (Juang et al., 2013). However, there are limitations to qRT-PCR and ChIP analysis when investigating the activity of odr-1 22G RNA. Neither technique offers a dynamic or cell-specific readout of odr-1 22G RNA function. Here, we show a fluorescent reporter that is capable of visualizing cell specific changes in odr-1 22G RNA in response to olfactory adaptation to the odor butanone. The fluorescence reporter that detects odr-1 22G RNA is inserted via Mos Single Copy Insertion (MosSCI) (Frøkjaer-Jensen et al., 2008). The sensor has the capability to detect specific odr-1 22G RNA in cells throughout the worm and specifically the germline. The creation of a single copy insertion strain that includes an odr-1 22G small RNA sensor allows us to test our hypothesis that a 22G small RNA-directed pathway is dynamically activated during olfactory adaptation.

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