Developments in RNA interference and genetic transformation to define gene function in parasitic helminths.

Advances in DNA sequencing technologies have led to the rapid accumulation of gene sequence data with whole genome sequences in draft form available for a number of important helminth parasites of animals andplants.There is an urgent requirement to establish reverse genetics tools to define the function of individual genes to enhance our knowledge of parasitism and to identify key parasite genes and their products which can provide the basis for novel control procedures be these new chemical interventions or vaccines. RNA interference (RNAi) is a reverse genetics technique which permits the gene-specific degradation of mRNA by the introduction of complementary double-stranded RNA (dsRNA). This has been achieved by a variety of approaches including soaking, electroporation or, in the case of Caenorhabditis elegans, by microinjection. The discovery that RNAi could be induced by simply soaking C. elegans the worm in dsRNA (Tabara et al. 1998) revolutionised the definition of gene function in this organism and encouraged the view that the procedure would be applied readily to parasitic helminths. Here, Zhuang and Hunter (2012) review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. They propose that these discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species. Following the discovery that RNAi could be induced by soaking, it was not long before the first report of successful RNAi in a parasitic nematode was published with Hussein et al. (2002) reporting a large diminution in secreted AChE from Nippostrongylus brasiliensis. Soaking adult worms in dsRNA for AChE A or B, both secreted acetylcholinesterases, resulted in knockdown of the homologous enzyme activity and that of two other closely related variants of the enzyme (Hussein et al. 2002). However, transcript knockdown could not be confirmed by RTPCR. Moreover, Hussein et al. outlined in Selkirk et al. (2012) failed to observe knockdown by RT-PCR of six other targets by soaking, these being homologues of ubiquitin, paramyosin (unc-15 in C. elegans), calponin (unc-87), protein disulphide isomerase 3 (PDI-3), phospholipase A2 (PLA2) and β-tubulin. This inconsistency of knockdown was emphasised by subsequent studies (Geldhof et al. 2007; Visser et al. 2006). Selkirk et al. (2012) discuss options which have been tested to enhance RNAi efficiency including feeding bacteria engineered to express dsRNA, methods to activate/enhance feeding, as well as the use of electroporation. The paper by Dalzell et al. (2012) addresses many of what the authors consider to be themost important aspects of RNAi experimental design in parasitic helminths. The paper suggests ways of standardising RNAi experiments to allow meaningful comparisons to be drawn between studies in different laboratories and in different organisms. They also address issues such as parasite culture, target gene selection, the use of dsRNA or siRNA as mediators of knockdown, delivery and, most importantly, target transcript quantification and adequate controls. Finally, they propose an experiment workflow to help standardise outputs from RNAi experiments and point out some of the potential pitfalls and solutions associated with various technical aspects. Zawadzki et al. (2012) discuss work which evaluated three different methods for introducing dsRNA into the sheep parasitic nematode Haemonchus contortus namely (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targeting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. They targeted five genes that are essential inCaenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). They describe reduction in transcript levels for each gene tested and, when delivery was by feeding, they noted observable changes 557