title: Integration of reporter transgenes into Schistosoma mansoni chromosomes 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.
authors.affiliation: Department of Tropical Medicine, and Biomedical Sciences Program, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
authors.forename: Kristine J
authors.initials: KJ
authors.lastname: Kines
authors.forename: Maria E
authors.initials: ME
authors.lastname: Morales
authors.forename: Victoria H
authors.initials: VH
authors.lastname: Mann
authors.forename: Geoffrey N
authors.initials: GN
authors.lastname: Gobert
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002875:Chromosomes / Q000235:genetics / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D017930:Genes, Reporter; D005822:Genetic Vectors; D049751:Genome, Helminth; D023281:Genomics; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000302:isolation & purification; D049409:Luciferases, Firefly / Q000235:genetics; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity / Q000821:virology; D017384:Sequence Deletion; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics; D016662:Virus Integration / Q000235:genetics
chemical_list: D000199:Actins; D017931:DNA Primers; D018509:DNA, Helminth; D004279:DNA, Viral; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D049409:Luciferases, Firefly