Pseudotyped murine leukemia virus for schistosome transgenesis: approaches, methods and perspectives.
Draft genome sequences for the human schistosomes, Schistosoma japonicum, S. mansoni and S. haematobium are now available. The schistosome genome contains ~11,000 protein encoding genes for which the functions of few are well understood. Nonetheless, the newly described gene products and novel non-coding RNAs represent potential intervention targets, and molecular tools are being developed to determine their importance. Over the past decade, noteworthy advances has been reported towards development of tools for gene manipulation of schistosomes, including gene expression perturbation by RNAi, and transient and stable transfection including transgenesis mediated by genome integration competent vectors. Retrovirus-mediated transgenesis is an established functional genomic approach for model species. It offers the means to establish gain- or loss-of-function phenotypes, supports vector-based RNA interference, and represents a powerful forward genetics tool for insertional mutagenesis. Murine leukemia virus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein mediates somatic transgenesis in S. mansoni, and vertical transmission of integrated transgenes in S. mansoni has been demonstrated, leading the establishment of transgenic lines. In addition, MLV transgenes encoding antibiotic resistance allow the selection of MLV-transduced parasites with the appropriate antibiotics. Here we describe detailed methods to produce and quantify pseudotyped MLV particles for use in transducing developmental stages of schistosomes. Approaches to analyze MLV-transduced schistosomes, including qPCR and high throughput approaches to verify and map genome integration of transgenes are also presented. We anticipate these tools should find utility in genetic investigations in other laboratories and for other helminth pathogens of important neglected tropical diseases.
Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, George Washington University, 2300 Eye Street NW, Washington, DC, 20037, USA.
Victoria H
VH
Mann
Sutas
S
Suttiprapa
Danielle E
DE
Skinner
Paul J
PJ
Brindley
Gabriel
G
Rinaldi
D000818:Animals; D018014:Gene Transfer Techniques; D016678:Genome; D005854:Germ Cells / Q000821:virology; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000472:pathogenicity; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D016254:Mutagenesis, Insertional; D012550:Schistosoma mansoni / Q000235:genetics*; D014161:Transduction, Genetic