Transgene driven RNAi for Cell-specific knock-down of gene function in targeted C. elegans neurons

In C. elegans the available approaches to identify the neuron(s) where the function of a gene is required for a given trait (e.g. developmental, behavioral, etc.) are time consuming and largely restricted to non essential genes for which mutants are available. We have described a simple reverse genetics approach for reducing, in chosen C. elegans neurons, the function of genes (Esposito et al., 2007). The method is based on the expression, under cell specific promoters, of sense and antisense RNA corresponding to a gene of interest. We do not clone the knock-down constructs but directly inject in worms a mixture of two separate PCR fusion reactions. In each fusion a promoter specifically active in the neuron(s) we want to target drives the transcription, either in the sense or in the antisense orientation, of a CDS fragment of the gene we want to knock-down. By targeting the Green Fluorescent Protein gene, gfp, and the genes osm-10 and osm-6, we showed that this approach leads to efficient, heritable and cell autonomous knock-downs of gene function, even in neurons usually refractory to classic RNA interference (RNAi). By targeting the essential and ubiquitously expressed gene, gpb-1, which encodes a G protein ?-subunit, we identified two distinct sets of neurons in which the function of gpb-1 is required to regulate two distinct behavior: egg-laying and avoidance of repellents. The cell specific knock-downs obtained with this approach provide information (is the gene necessary?) that is complementary to that provided by the cell specific rescue of loss of function mutations (is the gene sufficient?). We will present examples to show how this approach can be used: i) to address questions in neural development (kal-1); ii) to identify the neuron(s) of a circuit where the function of a gene is required for a given behavior (gpb-1); iii) to study the neuronal function of pleiotropic and/or essential genes (gpb-1, unc-70, smn-1). We will also discuss the limitations of the method and what we still do not know about the mechanism and the features of the gene function knock-down that this procedure elicits.