Nucleic acid transfection and transgenesis in parasitic nematodes

Abstract

SUMMARY Transgenesis is an essential tool for assessing gene function in any organism, and it is especially crucial for parasitic nematodes given the dwindling armamentarium of effective anthelmintics and the consequent need to validate essential molecular targets for new drugs and vaccines. Two of the major routes of gene delivery evaluated to date in parasitic nematodes, bombardment with DNA-coated microparticles and intragonadal microinjection of DNA constructs, draw upon experience with the free-living nematode Caenorhabditis elegans. Bombardment has been used to transiently transfect Ascaris suum, Brugia malayi and Litomosoides sigmodontis with both RNA and DNA. Microinjection has been used to achieve heritable transgenesis in Strongyloides stercoralis, S. ratti and Parastrongyloides trichosuri and for additional transient expression studies in B. malayi. A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developing B. malayi larvae and results in transgene inheritance through host and vector passage. Assembly of microinjected transgenes into multi-copy episomal arrays likely results in their transcriptional silencing in some parasitic nematodes. Methods such as transposon-mediated transgenesis that favour low-copy number chromosomal integration may remedy this impediment to establishing stable transgenic lines. In the future, stable transgenesis in parasitic nematodes could enable loss-of-function approaches by insertional mutagenesis, in situ expression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins.

The barber's pole worm CAP protein superfamily — A basis for fundamental discovery and biotechnology advances

“…The availability of the entire genomic and transcriptomic sequence data sets Laing et al, 2013) and advances in functional genomics (Lok, 2012) should open up opportunities for fundamental investigations of CAP proteins and their genes in different developmental stages of H contortus. The present transcription profiles for genes encoding CAP proteins suggest that these molecules play diverse but critical roles in development and reproduction at different life cycle stages of this parasite.…”

“…For instance, virus-based transduction (Hagen et al, 2014;Hagen et al, 2015a,b) might be useful for the delivery of microRNA-adapted small hairpin RNAs (shRNAmirs) into H. contortus to achieve effective gene silencing. This area deserves attention and has the potential to overcome the inefficiency of RNAi approaches employed to date on H. contortus (reviewed by Knox et al, 2007;Lok, 2012). The development of an effective gene-silencing system for H. contortus could lead to a major and rapid advance of our understanding of orphan CAP proteins and their genes, and could help establish their actual involvement in biological and developmental pathways in this important parasite as well as parasite-host interactions at the immuno-molecular level.…”

A perspective on genomic-guided anthelmintic discovery and repurposing using Haemonchus contortus

“…The goal is to identify genes or molecules whose inactivation by one or more drugs will selectively kill parasites but not harm their host(s). As most parasitic worms, including H. contortus, are difficult to produce and maintain without their host animal and functional genomic studies have been very challenging (reviewed by Geldhof et al, 2007;Knox et al, 2007;Lok, 2012), gene essentiality can be inferred from non-wild-type knock-down information (e.g., lethality) in C. elegans and/or other model organisms (Zhong and Sternberg, 2006;Lee et al, 2008). Using extensive genomic and transcriptomic resources now available for H. contortus (see Liang et al, 2013;Schwarz et al, 2013), druggable molecules can be predicted based on essentiality predictions, ensuring gene transcription in the parasitic stages of the parasite.…”

Cracking the nodule worm code advances knowledge of parasite biology and biotechnology to tackle major diseases of livestock

“…Interestingly, according to the present analysis, only two of four RNA-induced silencing complex (RISC) proteins and 11 of 28 argonaute proteins of C. elegans have orthologs in O. dentatum , indicating substantial differences between these two nematodes and unique silencing pathways within each nematode. In spite of these differences and challenges of conducting RNAi in parasitic nematodes (Geldhof et al, 2007; Knox et al, 2007; Lok, 2012), there seems to be a prospect of undertaking targeted functional genomic experiments in O. dentatum , possibly using virus-based transduction, given that this worm can be maintained in vitro through several moults (Daugschies and Watzel, 1999). …”