Optimization of RNAi efficiency in PVD neuron of C. elegans

Abstract PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to the elaborate and stereotype dendrite morphology in this neuron. The molecular basis for dendrite maintenance and regeneration is poorly understood. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans . However, the feeding method often fails when it comes to nervous system. Using an optimal induction condition for the dsRNA expression in E coli , we fed the worm strains with HT115 bacteria expressing dsRNA against genes like mec-3, hpo-30, and tiam-1 , whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the strains such as nre-1(-) lin-15b(-), lin-15b(-) and sid-1(-); lin-15b(-); Punc-119::sid-1[+] resulted in significant reduction of dendrite branching. However, the phenotypes were significantly modest compared to the respective loss of function mutants. To obtain stronger phenotype for PVD specific genes, we have made a strain, which strongly expresses sid-1 under mec-3 promoter specific for PVD. When Pmec-3::sid-1 is expressed in either nre-1(-);lin-15b(-) or lin-15b(-) background, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to nre-1(-);lin-15b(-) and lin-15b(-) background alone. Next we tested the nre-1(-) lin-15b(-), P mec-3-sid-1[+] strain for the knockdown of genes playing role in dendrite regeneration process. We found that when aff-1 and ced-10 genes were knocked down in the nre-1(-) lin-15b(-), P mec-3-sid-1[+] background, the dendrite regeneration was significantly reduced and the extent of reduction was comparable to that of the mutants of aff-1 and ced-10 . Essentially, our strain expressing sid-1 in PVD neuron optimizes the condition for RNAi for high throughput screening for PVD development, maintenance and regeneration.