title: An antibiotic selection marker for schistosome transgenesis.
abstract: Drug selection is widely used in transgene studies of microbial pathogens, mammalian cell and plant cell lines. Drug selection of transgenic schistosomes would be desirable to provide a means to enrich for populations of transgenic worms. We adapted murine leukaemia retrovirus vectors - widely used in human gene therapy research - to transduce schistosomes, leading to integration of transgenes into the genome of the blood fluke. A dose-response kill curve and lethal G418 (geneticin) concentrations were established: 125-1,000μg/ml G418 were progressively more toxic for schistosomules of Schistosoma mansoni with toxicity increasing with antibiotic concentration and with duration of exposure. By day 6 of exposure to ⩾500μg/ml, significantly fewer worms survived compared with non-exposed controls and by day 8, significantly fewer worms survived than controls at ⩾250μg/ml G418. When schistosomules were transduced with murine leukaemia retrovirus encoding the neomycin resistance (neoR) transgene and cultured in media containing G418, the neoR transgene rescued transgenic schistosomules from the antibiotic; by day 4 in 1,000μg/ml and by day 8 in 500μg/ml G418, significantly more transgenic worms survived the toxic effects of the antibiotic. More copies of neoR were detected per nanogram of genomic DNA from populations of transgenic schistosomes cultured in G418 than from transgenic schistosomes cultured without G418. This trend was G418 dose-dependent, demonstrating enrichment of transgenic worms from among the schistosomules exposed to virions. Furthermore, higher expression of neoR was detected in transgenic schistosomes cultured in the presence of G418 than in transgenic worms cultured without antibiotic. The availability of antibiotic selection can be expected to enhance progress with functional genomics research on the helminth parasites responsible for major neglected tropical diseases.
authors.affiliation: Department of Microbiology, Immunology and Tropical Medicine, School of Medicine & Health Sciences, The George Washington University, 2300 I Street NW, Washington, DC 20037, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rinaldi
authors.forename: Sutas
authors.initials: S
authors.lastname: Suttiprapa
authors.forename: José F
authors.initials: JF
authors.lastname: Tort
authors.forename: Anne E
authors.initials: AE
authors.lastname: Folley
authors.forename: Danielle E
authors.initials: DE
authors.lastname: Skinner
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
mesh_terms: D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D018014:Gene Transfer Techniques; D005839:Gentamicins / Q000494:pharmacology*; D008967:Molecular Biology / Q000379:methods*; D030781:Organisms, Genetically Modified; D012190:Retroviridae / Q000235:genetics; D012550:Schistosoma mansoni / Q000235:genetics*; D012641:Selection, Genetic
chemical_list: D000871:Anthelmintics; D003470:Culture Media; D005839:Gentamicins; C010680:antibiotic G 418