Using piRNA interference to quantify acute and inherited silencing in Caenorhabditis elegans with no requirement for genetic crosses: a case study.

Abstract We recently developed a piRNA-based silencing assay called piRNA interference (piRNAi), where acute gene silencing is mediated by synthetic piRNAs expressed from extra-chromosomal array. Arrays are spontaneously lost every generation which can be used to quantify the persistence of silencing in the absence of a silencing trigger piRNAs. The assay allows inheritance assays by injecting piRNAs directly into mutant animals and targeting endogenous genes (e.g., him-5 and him-8) with obvious phenotypes (increased male frequency). In some cases this approach is advantageous because genetic crosses, especially for transgenes that are unpaired during meiosis, can have a large influence on epigenetic silencing. Here we demonstrate the use of the piRNAi assay by silencing in the ribonucleotidyltransferase rde-3 (ne3370) mutant and quantifying acute and inherited silencing. We find that in the absence of rde-3, acute silencing is reduced but still detectable, whereas we see no evidence of inherited silencing.