Quantitative retrotransposon anchored PCR confirms transduction efficiency of transgenes in adult Schistosoma mansoni

Gabriel Rinaldi*, Sutas Suttiprapa, Paul J. Brindley

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Citations (SciVal)

Abstract

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.

Original languageEnglish
Pages (from-to)70-76
Number of pages7
JournalMolecular and Biochemical Parasitology
Volume177
Issue number1
Early online date18 Jan 2011
DOIs
Publication statusPublished - 31 May 2011
Externally publishedYes

Keywords

  • Anchored PCR
  • Integration
  • Luciferase
  • Pseudotyped retrovirus
  • Retrotransposon
  • Schistosome
  • Transgene

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