Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

Abstract

Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

Author Summary

Parasitic roundworms sicken and debilitate over one billion people, most of whom subsist on less than two US dollars per day. There are no vaccines and few drugs available to treat and prevent these infections. Basic research leading to new therapies has been hampered because we lack methods to study gene function in parasitic roundworms. One such method is transgenesis, a process by which gene function is inferred by studying the effects of transferring native or altered copies of genes into subject organisms. Our laboratory has developed a system for transferring synthetic genes into parasitic roundworms of the genus Strongyloides and for obtaining temporary expression of these “transgenes”. Until now, however, we have been unable to propagate these transgenic parasites in the laboratory. This paper describes a new technique that allows us to establish and maintain self-perpetuating lines of transgenic parasites for study. This represents a fundamental advance in the methodology for studying gene function in parasitic roundworms and should greatly facilitate the discovery of new therapies.