| Despite more than a century of control efforts, schistosomiasis remains a major public health problem in more than 75 countries with over 200 million people currently infected. In addition to the morbidity and mortality, this neglected tropical disease has an enormous impact due to the debilitating effects of chronic morbidity and the ability to impair childhood growth, intellectual development, pregnancy outcomes, and economic performance. Recent progress has advanced the field of helminth genomics. However, the gene manipulation tools that would allow functional analysis of sequences from these neglected tropical disease pathogens are not well developed or routine. Retroviral-mediated transduction offers a potential means to establish transgenic lines of schistosomes, to elucidate gene function and expression, and to advance functional genomics for schistosomes and other parasitic helminths. At the outset of this project, my colleagues and I hypothesized that developmental stages of Schistosoma mansoni can be transduced using the Moloney Murine Leukemia Retrovirus (MLV), pseudotyped with Vesicular Stomatitis Virus Glycoprotein (VSVG) and that this strategy can be employed to develop transgenic schistosomes. We hypothesized that the pseudotyped glycoprotein would allow the retrovirus to bind to the schistosome tegument, followed by integration of the provirus into the chromosomes. It was also hypothesized that VSVG-MLV could mediate somatic and, thereafter, heritable germ line transgenesis in S. mansoni.;The VSVG-MLV retroviral vector was modified to include reporter genes and endogenous schistosome promoters, and employed for the transduction of schistosome developmental stages. The interaction of the retrovirus and parasite tegument was investigated by immunolocalization approaches, and the presence of the reporter transgene was confirmed by Southern hybridization analysis and reverse transcription-polymerase chain reaction (RT-PCR). Analysis of the integration of reporter transgenes into S. mansoni revealed probable widespread integration into the genome. The integration junctions of the provirus into schistosome chromosomes were recovered using an anchored PCR-based approach, and sequenced to confirm somatic transgenesis. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. This, along with the absence of a 2 bp deletion from the terminus of the integrated 5'-LTR, appeared to differentiate the phenomenon of proviral integration in schistosome chromosomes from the mammalian paradigm. Reporter transgene expression driven by the schistosome actin gene promoter was demonstrated, and appeared to be developmentally expressed in the schistosomule stage.;To explore germ line transgenesis of schistosomes, eggs and larval stages were transduced by VSVG-MLV and introduced back into the life cycle by infection of both the snail and the mammalian hosts. Vertical transmission was confirmed in the progeny cercariae -- derived by asexual reproduction after infection of snails with transgenic miracidia. The presence of transgenes in the progeny cercariae was detected by PCR specific for proviral transgenes and by immunolocalization of luciferase reporter protein.;In overview, these findings indicated the heritable transmission of retroviral transgenes vertically from the egg, through the miracidia, sporocyst, and into the cercarial stage of the schistosome. In addition to the novel demonstration of transduction of schistosomes by pseudotyped retrovirus, these studies also demonstrated that square wave electroporation can be employed to transduce developmental stages of schistosomes with pseudotyped retroviruses. Together, the findings presented in this dissertation indicated the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, and provided evidence - for the first time - of vertical transmission of an integrated transgene in schistosomes through germ line transgenesis. |
