RNAi Gene knockdown of R-opsin and F-actin in the marineannelid Streblospio benedicti by delivering dsRNA

Abstract

BackgroundRNA interference (RNAi) is a genetic tool to disrupt the expression of selected genes by delivering dsRNA into a specific tissue of an organism, resulting in a gene knockdown. We apply RNAi methods to the marine polychaete Streblospio benedicti, which is a model system for studying evolutionary and developmental biology, which currently has no established methods for gene expression manipulation. ResultsHere we describe a RNAi gene knockdown methodology using two different approaches depending on developmental stage. We feed bacteria expressing dsRNA to early swimming larvae and microinject in vitro transcribed dsRNA in juveniles. We used two genes for testing: R-opsin and F-actin. For both developmental stages, gene knockdown was validated using qPCR (quantitative Real-Time PCR). For larvae, we also visualized reduction in RNA expression using Hybridization Chain Reaction (HCR) in situ hybridization. ConclusionsWe show that both larval feeding and juvenile microinjections are sufficient delivery mechanisms for dsRNA to result in a RNAi gene knockdown. The efficiency can vary depending on the gene and timing, but this is typical for RNAi based approaches.