Silencing Specific Paramecium tetraurelia Genes by Feeding Double-Stranded RNA

INTRODUCTION This protocol describes an easy and efficient method to knock down gene expression in a homology-dependent manner by feeding Paramecium with bacteria-producing double-stranded (ds) RNA corresponding to a portion of the target gene. The target sequence is cloned in a plasmid between two convergent T7 promoters (the L4440 plasmid) and used to transform an E. coli strain devoid of RNase III activity and expressing the T7 polymerase gene under the control of an inducible Plac promoter (strain HT115-DE3). Inserts corresponding to ~400 bp of the coding sequence to be silenced are recommended; very short inserts (<100 bp) have variable silencing efficiencies. As a rule, pairs of paralogs resulting from the most recent whole-genome duplication are so similar in sequence that dsRNA from one paralog will cosilence both. Although the level of sequence identity required has not been explored fully, more divergent paralogs can occasionally be co-silenced too. When Paramecium tetraurelia cells feed on these bacteria, dsRNA reaches the cytoplasm through unknown mechanisms and triggers a potent RNA interference (RNAi) response, resulting in the degradation of the targeted mRNA. The protocol generally gives a visible phenotype in 24-48 h, depending on the targeted gene, a delay which might correspond to the time necessary for the pre-existing protein stock to be diluted by cell growth. Silencing induced in this manner is only post-transcriptional; when cells are transferred back to normal medium bacterized with Klebsiella pneumoniae, the phenotype usually reverts to wild type within a few cell divisions.