We have developed a method to derive transgenic schistosomes that utilizes the murine leukemia virus (MLV) to transfect schistosome eggs. After infecting snails with miracidia from eggs transduced by the MLV retrovirus, genomic DNA from cercariae released from the snails revealed the presence of transgenes, demonstrating that retroviral transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. Transgenic cercariae could be cryopreserved and retained infectivity for mice, and were in turn transmitted through the sexual developmental (meiosis) cycle to produce F1 generations of transgenic schistosomes. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. These findings provided the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Here we propose to enhance our successful approach to schistosome transgenesis by the addition of antibiotic selection of transgenic schistosomes. Drug selection is widely used in transgene studies of microbial pathogens, mammalian cell and plant cell lines. Drug selection of transgenic schistosomes would provide a means to enrich for populations of transgenic worms. Recently we have demonstrated that MLV-transduced schistosomules expressing a neomycin resistance marker could be rescued on the aminoglycoside antibiotic, geneticin (G418). We hypothesize, based on these preliminary findings, that transgenic lines of schistosomes can be established by transducing the zygote within the schistosome eggshell and subsequently infecting snails with the resulting miracidia using concurrent selection on antibiotics to rescue transgenic worms and eliminating wild type worms or transgenic worms not expressing the drug resistance maker. Our three specific aims are:
Aim 1. Investigate sensitivity of Schistosoma mansoni eggs to three discrete classes of antibiotics: (1) aminoglycosides e.g. G418;(2) aminonucleosides e.g. puromycin;3) glycopeptides e.g. zeocin - for which selectable markers for all three categories are readily available.
Aim 2. Optimization of antibiotic resistance gene expression in retrovirus-transduced and transgenic schistosomes.
Aim 3. Antibiotic selection of retrovirus-transduced schistosome developmental stages, in particular in vitro laid eggs, on these antibiotics. The availability of antibiotic selection can be expected to enhance functional genomics and research progress on helminth parasites responsible for major neglected tropical diseases.
Schistosomes, or blood flukes, are responsible a type of parasitic disease known as schistosomiasis which afflicts >200 million people in impoverished regions of the developing world. The genome sequence of these parasitic worms has been decoded recently. The studies proposed deal with genetically modified pathogens, in particular schistosome transgenic lines expressing antibiotic resistance markers to determine the importance of newly described genes;we anticipate that the information obtained can lead to new interventions for this neglected tropical disease.