Abstract
Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.
Highlights
The analysis of gene function has been restricted to a few model systems because techniques for manipulating gene expression in one species are often not transferable to other species
We used inbred strains of the free-living nematodes Auanema rhodensis APS4, A. freiburgensis APS710, and Pristionchus pacificus PS312 derivative “97”40. These strains were maintained in the laboratory on nematode growth medium (NGM) agar plates seeded with the streptomycin-resistant Escherichia coli OP50-1 strain and cultured at 20 °C by standard procedures for C. elegans[41]
Easy-to-score phenotypes when targeted by RNA interference (RNAi) in C. elegans[15,56]
Summary
The analysis of gene function has been restricted to a few model systems because techniques for manipulating gene expression in one species are often not transferable to other species. Recent technological advances in high-throughput sequencing, gene silencing strategies, and genome editing have changed this and opened the door to mechanistic studies in non-model systems. In a range of nematodes the RNAi response can be improved via the addition of chemicals (e.g., serotonin, octopamine, spermidine, lipofectamine) that enhance uptake of the dsRNA/siRNA from the medium[26,27,28,29,30]. Another option is the direct injection of dsRNA into the body cavity or gonad[22,31,32]. The addition of a substance that effectively spreads RNA from the site of injection can help to overcome the limitations of a less robust spreading system
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