In the analyses of multifunctional genes such as Ras which plays different roles in various tissues at different stages, the effect of these genes knockdown must be controlled strictly to prevent critical secondary effects such as lethality. Recently, cell-specific or time-specific RNAi is currently available. However, the conventional RNAi methods are insufficient for the analysis of the multifunctional genes.
We therefore established a novel method enables to simultaneously perform time- and cell-specific RNAi (T.C.RNAi). In T.C.RNAi, the cell-specific promoter which drives the cell-specific and constitutive expression and the heat shock promoter which drives the time-specific and global expression are adopted. The one RNA strand is driven by the cell-specific promoter, and the other one is done by the heat shock promoter, leading to drive the expression of double stranded RNA only in the targeted cell. To confirm the effect of T.C.RNAi, we performed the GFP knockdown. The effect of knockdown by T.C.RNAi appeared only in a targeted cell after heat shock treatment, indicating T.C.RNAi is capable of simultaneous time- and cell-specific knockdown.
Using T.C.RNAi, functions of Let-60Ras in exploratory behavior was analyzed. We found Let-60Ras mutants showed abnormal locomotion behavior where they continue to move in a circled pattern under a circumstance far from food resources. Because of the abnormal and biased head movement in the Let-60Ras mutant, we focused on the foraging behavior in which animals show exploratory and constitutive head movement on locomotion. Foraging behavior is known to be regulated by a neural circuit composed of IL1, OLQ, and RMD neurons. The cell-specific knockdown of Let-60Ras and rescue experiments showed Let-60Ras functions in the neural circuit to control exploratory behavior. In addition, the knockdown of Let-60Ras in RMD motor neurons induced mislocalization of glutamate receptors GLR-1. To reveal the temporal profile of Let-60Ras in RMD neurons, we performed conditional knockdown of Let-60Ras by T.C.RNAi. The knockdown of Let-60Ras at the adult stage induced GLR-1 mislocalization. Further, we observed the knockdown of Let-60Ras at the adult stage caused loopy-patterned locomotion. These results suggest Let-60Ras functions in RMD motor neurons at the adult stage to regulate GLR-1 localization for the modulation of exploratory behavior.
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