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 leukemia retrovirus (MLV) 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 to 1,000 μg/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 ≥500 μg/ml, significantly fewer worms survived compared with non-exposed controls and by day 8, significantly fewer worms survived than controls at ≥250 μg/ml G418. When schistosomules were transduced with MLV 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 1,000 μg/ml and by day 8 in 500 μg/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.

Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins

“…The images were captured consecutively, at 10 frames per second. In some experiments, samples of the schistosomes were removed from the culture and incubated in the fluorophores fluorescein diacetate (FDA), at 2.0 μg/ml, which stains live schistosomes, and propidium iodide (PI), at 0.5 μg/ml, which stains dead schistosomes, using previously reported methods (Peak et al, 2010; Rinaldi et al, 2012). In order to assess the effect of FCF on the F-actin structure, cercariae exposed to 500 μM FCF for 3 h were fixed in 4% paraformaldehyde for 1 h at 4 °C.…”

Developmental Sensitivity in Schistosoma mansoni to Puromycin To Establish Drug Selection of Transgenic Schistosomes

“…Previously, we reported that schistosomula expressing NeoR could be partially rescued and survived longer than control wild-type schistosomula when cultured in the presence of the aminoglycoside neomycin (55). Moreover, a germ line transgenic progeny (schistosomula), i.e., transgenic schistosomula obtained from snails infected with miracidia that had been hatched from MLV-transduced eggs, also exhibited resistance to G418 (22).…”

Suppression of Ov-grn-1 encoding granulin of Opisthorchis viverrini inhibits proliferation of biliary epithelial cells

“…Viability of flukes was determined on day 5 by observing movement of oral or ventral suckers using light microscopy in the presence of the vital dye, Trypan blue at 0.1% in PBS for 10 min at 37°C, 5% CO2. Flukes were scored as dead if the worm stained blue, or as alive if not stained or stained weakly with Trypan blue and the oral or ventral suckers was still moving (alive), or dead, if no movement was apparent during observation for 10 min duration (Gold, 1997, Rinaldi et al, 2012). Worm survival among treatment groups was compared using independent t- tests.…”