Nucleic acid transfection and transgenesis in parasitic nematodes

Abstract

SUMMARYTransgenesis 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 nematodeCaenorhabditis elegans. Bombardment has been used to transiently transfectAscaris suum, Brugia malayiandLitomosoides sigmodontiswith both RNA and DNA. Microinjection has been used to achieve heritable transgenesis inStrongyloides stercoralis, S. rattiandParastrongyloides trichosuriand for additional transient expression studies inB. malayi.A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developingB. malayilarvae 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 situexpression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins.