Optimization of RNAi efficiency in PVD neuron ofC. elegans

Abstract

AbstractPVD neuron ofC. eleganshas 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 feedingE. coliexpressing dsRNA has been the basis of several genome wide screens performed usingC. elegans. However, the feeding method often fails when it comes to nervous system. Using an optimal induction condition for the dsRNA expression inE coli, we fed the worm strains with HT115 bacteria expressing dsRNA against genes likemec-3, hpo-30,andtiam-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 asnre-1(-) lin-15b(-), lin-15b(-)andsid-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 expressessid-1undermec-3promoter specific for PVD. WhenPmec-3::sid-1is expressed in eithernre-1(-);lin-15b(-)orlin-15b(-)background, the higher order branching phenotype after RNAi ofmec-3, hpo-30,andtiam-1was significantly enhanced as compared tonre-1(-);lin-15b(-)andlin-15b(-)background alone. Next we tested thenre-1(-) lin-15b(-),Pmec-3-sid-1[+]strain for the knockdown of genes playing role in dendrite regeneration process. We found that whenaff-1andced-10genes were knocked down in thenre-1(-) lin-15b(-),Pmec-3-sid-1[+]background, the dendrite regeneration was significantly reduced and the extent of reduction was comparable to that of the mutants ofaff-1andced-10. Essentially, our strain expressingsid-1in PVD neuron optimizes the condition for RNAi for high throughput screening for PVD development, maintenance and regeneration.