Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.
Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.
Kines
Kristine J
KJ
Department of Tropical Medicine and Center for Infectious Diseases, Tulane University, Health Sciences Center, New Orleans, LA 70112, USA.
Mann
Victoria H
VH
Morales
Maria E
ME
Shelby
Bryan D
BD
Kalinna
Bernd H
BH
Gobert
Geoffrey N
GN
Chirgwin
Sharon R
SR
Brindley
Paul J
PJ
D000818:Animals; D001700:Biomphalaria; D015139:Blotting, Southern; D005455:Fluorescent Antibody Technique / Q000379:methods; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000235:genetics* / Q000502:physiology; D014161:Transduction, Genetic / Q000379:methods*; D014721:Vesicular stomatitis Indiana virus / Q000737:chemistry / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000235:genetics*; D016662:Virus Integration
D014760:Viral Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases