Integration of reporter transgenes intoSchistosoma mansonichromosomes mediated by pseudotyped murine leukemia virus

Abstract

The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus-mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG-pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR-based approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward toward germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes.

Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni

“…We have previously demonstrated that replication-incompetent Moloney murine leukaemia virus (MMLV) virions pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) were capable of transducing schistosomes and integrating into the schistosome genome [18,19]. After exposure of schistosomes to virions, harvested from producer cell cultures, immunofluorescence studies indicated that the VSVG envelope on the pseudotyped virions interacted with the schistosome surface after which the retroviral capsid and RNA genome was released within the surface cells [18].…”

“…The schistosome lifecycle was maintained in our laboratory by passaging through BALB/c mice as previously described [20]. Adult schistosomes, perfused from mice 49 days after infection with cercariae, were cultured at 37°C and 5% CO 2 in DMEM (Gibco), supplemented with 10% foetal bovine serum, 100 U penicillin and streptomycin, and small quantities of washed mouse erythrocytes, 2 ll washed erythrocytes (50% packed red cells)/ml culture medium, before and after exposure to virus [19].…”

“…We have recently described the use of pseudotyped Moloney murine leukaemia virus to accomplish transformation of S. mansoni [18,19]. Here, we report the design of a retroviral vector expressing a dsRNA hairpin loop, thus coupling a powerful delivery vehicle with a potent RNA interference (RNAi) mechanism to specifically silence expression of SmCB1.…”

Biogenic amines and the control of neuromuscular signaling in schistosomes

“…As an alternative method, researchers are beginning to use vector-based delivery of short hairpin RNAs (shRNAs), which in principle has the potential to deliver more sustained RNAi silencing in vivo. This technology builds on recent advances in the use of retroviral promoters for schistosome transgenesis (Kines et al 2008) and was recently adapted to RNAi silencing in S. mansoni with promising results (Tchoubrieva et al 2010). Additionally, in vivo gene silencing may be performed directly in the infected mouse by systemic intravenous delivery of parasite-specific siRNAs or morpholinos.…”

Discovering New Schistosome Drug Targets: The Role of Transcriptomics

“…Both Cathepsins B and D have been successfully targeted by RNAi in S. mansoni leading to reduced parasite growth in culture [43,44] and resulted in a fatal phenotype when parasites were returned to the mammalian host [44]. It is anticipated that transgenic schistosomes may be available in the near future especially in light of the recent publications presenting the integration of exogenous reporter genes into the S. mansoni genome [42,83], which may further facilitate drug discovery and testing.…”