Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

Frédéric Landmann,Barton E Slatko,William Sullivan,Jeremy M Foster

doi:10.1186/1756-3305-5-16

Frédéric Landmann, Barton E Slatko + Show 2 more

Open Access

https://doi.org/10.1186/1756-3305-5-16

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Abstract

BackgroundRNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans.ResultsWe report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week.ConclusionsWe observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.

Highlights

RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes
To test RNAi in B. malayi, we decided to target orthologs of a number of genes known to be involved in C. elegans development and which show clear cellular phenotypes when depleted in that species
A 2 day incubation with 100 nM heterogeneous short interfering RNA (hsiRNA) against B. malayi g-tubulin lead to cytokinesis defects during early embryogenesis, as visualized by our optimized immunostaining procedures (Figure 1), and was sufficient to target all zygotic and very early embryonic divisions

Summary

Introduction

RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. Since almost all filarial species that cause disease in humans rely on the bacterial endosymbiont Wolbachia for proper embryogenesis, development and viability, these symbionts have become a major drug target. While reverse genetic tools such as RNA interference (RNAi) are routinely used in C. elegans research to characterize gene function [12], RNAi experiments in animal parasitic nematodes have proven notoriously challenging [13,14,15,16,17,18] with few successes reported. There are a few reports of successful RNAi in filarial nematodes [20,21,22,23,24,25,26] several of these studies targeted the same genes. Various explanations for the limited success of RNAi in animal parasitic nematodes have been proposed [15,19,27]

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