Molecular & Biochemical Parasitology 129 (2003) 41–51 Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi  A. Aziz Aboobaker, Mark L. Blaxter ∗ Institute of Cell, Animal and Population Biology, Kings Buildings, University of Edinburgh, Edinburgh, EH9 3JT UK Received 28 January 2003; received in revised form 26 March 2003; accepted 26 March 2003 Abstract We describe the successful use of the reverse genetic technique RNA interference (RNAi) to investigate gene function in the human filarial nematode parasite Brugia malayi. We used fluorescently labelled double stranded RNA (dsRNA) to demonstrate that 300 bp molecules are able to enter adult females in culture while they remain excluded from microfilariae (mf). We have developed an optimised microvolume culture system to allow the exposure of parasites to high concentrations of dsRNA for extended periods. Culturing of adult female parasites in this system for 24 h does not significantly reduce parasite lifespan or mf release in culture. Three B. malayi genes, -tubulin (Bm-tub-1), RNA polymerase II large subunit (Bm-ama-1) and B. malayi mf sheath protein 1/mf22 (Bm-shp-1) were targeted by soaking adult female B. malayi in dsRNA complementary to these transcripts in the optimised culture system. Targeting of the two housekeeping genes Bm-tub-1 and Bm-ama-1 led to a reduction in the levels of their transcripts, as assessed by reverse transcriptase coupled PCR (RT-PCR), and resulted in parasite death in culture. In contrast, targeting of the Bm-shp-1 gene was not lethal to adult females in culture. A marked reduction in mf release was observed for shp-1 RNAi parasites compared to controls and in addition 50% of mf released did not have fully elongated sheaths. This “short” phenotype correlated with the loss of the stockpiled shp-1 transcript from developing mf in treated adult female gonads. From these data we conclude that RNAi may be a useful method for assessment of drug target potential of genes identified in filarial gene discovery projects. © 2003 Elsevier Science B.V. All rights reserved. Keywords: RNAi; Brugia malayi; Caenorhabditis elegans; Filarial nematode; Phenotype; Gene function 1. Introduction For model, genetically tractable organisms such as the free-living nematode Caenorhabditis elegans [1], an ar- ray of genetic methods have been developed that permit hypothesis-driven investigation of function starting from sequence [2–4]. Many of these techniques, such as trans- genesis [5], directed mutation [6] or gene replacement, are inappropriate for parasitic species with complex life- cycles, obligatory outcrossing and large brood sizes. Par- asitic nematode genome projects, inspired by the success of the C. elegans program, have generated sequences for Abbreviations: mf, microfilariae; RNAi, RNA interference; FITC, flu- orescein isothiocyanate; dsRNA, double stranded RNA; RT-PCR, reverse transcriptase coupled PCR  Note: DNA sequences reported in this paper have been deposited in the GenBank database with accession numbers Bm-tub-1 (BMA551180) and Bm-ama-1 (BMA551181). ∗ Corresponding author. Tel.: +44-131-650-6760; fax: +44-131-650-7489. E-mail address: mark.blaxter@ed.ac.uk (M.L. Blaxter). tens of thousands of genes [7–10]. Many of these genes have proved to be hard to annotate with functional de- scriptors, because they have limited similarity to genes from well-studied organisms. Even when genes can be annotated robustly through homology, it has been diffi- cult to prove essential function in living parasites. We are interested in developing informatic and experimental methods for the identification of potential targets in ne- matodes that are only distantly related to the C. elegans model. A new reverse genetic technology, based on the discov- ery that double stranded RNA (dsRNA) molecules derived from the same sequence as a targeted mRNA can result in specific and significant enzymatic degradation of the mRNA [11], has the promise of overcoming the current impasse in functional analysis of unknown genes [12]. This phenomenon, termed RNA interference (RNAi), was first experimentally demonstrated in C. elegans [13], but plays a role in many previously described gene silencing phenomena, and appears to be based on hijacking of a conserved cellular machinery that normally has a role in 0166-6851/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0166-6851(03)00092-6