Transformation in parasitic nematodes at last?

Abstract

It is somewhat ironic that nematodes, represented by Caenorhabditis elegans, have been at the forefront of genetic and genomic research for the past 20 years, culminating with the completion of the genomic sequence in 1998. This was the first complete multicellular eukaryote sequence and, of greater general significance perhaps, served as the test-bed for the development of large-scale sequencing technology. However, despite this tremendous basic resource and a rapidly expanding parasitic nematode expressed sequence tag (EST) dataset (http://www.nematode.net), parasitic nematodes have lagged far behind other parasite genera in recent years owing to a lack of molecular tools for functional genomic analysis.The report by Lok and Massey [1xTransgene expression in Strongyloides stercoralis following gonadal microinjection of DNA constructs. Lok, J.B. and Massey, H.C. Mol. Biochem. Parasitol. 2002; 119: 279–284Crossref | PubMed | Scopus (30)See all References[1], the expression of a green fluorescent reporter (GFP) transgene in developing eggs of Strongyloides stercoralis females microinjected with a GFP reporter construct, is a sign of considerable progress. This provides us with several reasons for optimism. First, the use of S. stercoralis is an important development because this parasite provides access to at least one free-living generation and the potential of recovering the first putatively transgenic progeny without the need to infect a host. This removes one of the most challenging barriers to functional genomics in parasitic nematodes of mammals: the near absolute necessity to infect a host to produce the next generation. It is no coincidence that the parasitic protozoa yielded to transgenesis after in vitro culture was made possible. Unfortunately, the apparently transgenic eggs that showed GFP expression during early in utero development failed to hatch, presumably as a result of a lethality associated with the observed high levels of GFP expression. Therefore, if truly heritable transformation is to be achieved (as it is clear from Caenorhabditis elegans that the majority of transformation events following microinjection are not inherited), obtaining a transgenic line or strain will be a numbers game. The high frequency of apparently transgenic eggs observed suggests that the necessary numbers will also be available if the reporter-associated lethality can be overcome, and the ability to culture the injected free-living or heterogonic females in vitro means that it will be possible to recover and screen every viable offspring.Second, the authors used endogenous promoters from highly expressed S. stercoralis genes to drive reporter gene expression. The promoter sequences were isolated using complementary DNA (cDNA) sequence as a starting point. The availability of many thousands of parasite ESTs suggests that obtaining other promoters will not be a limiting factor in the development of the technologyFinally, the promising results reported here will, hopefully, encourage other labs (perhaps using other species with in vitro culture potential) and the relevant funding agencies to take up the challenge of developing a system for generating truly heritable transgenesis. It is clearly a feasible goal and one that is worth pursuing.