Short technical reportQuantitative retrotransposon anchored PCR confirms transduction efficiency of transgenes in adult Schistosoma mansoni

A quantitative retrotransposon anchored PCR (qRAP) that utilizes endogenous retrotransposons as a chromosomal anchor was developed to investigate integration of transgenes in Schistosoma mansoni. The qRAP technique, which builds on earlier techniques, (i) Alu-PCR which has been used to quantify lentiviral (HIV-1) proviral insertions in human chromosomes and (ii) a non-quantitative retrotransposon anchored PCR known to detect the presence of transgenes in the S. mansoni genome, was tested here in a model comparison of retrovirus-transduced adult schistosomes in which one group included intact worms, the other included fragments of adult worms. At the outset, after transducing intact and viable fragments of schistosomes with reporter RNAs, we observed more reporter activity in fragments of worms than in intact worms. We considered this simply reflects the increased surface area in fragments compared to intact worms exposed to the exogenous reporter genes. Subsequently, intact worms and worm fragments were transduced with pseudotyped virions. Transgene integration events in genomic DNA extracted from the virion-exposed worms and worm fragments were quantified by the qRAP, which revealed that fragmenting adult schistosomes resulted in increased density of proviral integrations. The qRAP findings confirmed the likely value of this qRAP technique for quantification of transgenes integrated in schistosome chromosomes. Last, considering the absence of schistosome cell or tissue lines, primary culture of fragmented worms offers an opportunity to optimize transgenesis, and other functional genomic approaches.

A quantitative retrotransposon-anchored PCR (qRAP) was developed to quantify integration events in retrovirus-transduced Schistosoma mansoni. Transgene copy number estimated from fragmented parasites was significantly higher than in intact worms.184Research highlights► We developed a quantitative-Retrotransposon-Anchored PCR (qRAP) for schistosomes. ► qRAP estimates transgene integration events in the schistosome genome. ► Reporter RNAs indicated that dicing adult worms improved transduction efficiency. ► qRAP confirmed increased transgene integration density in diced worms.