piggyBactransposon mediated transgenesis of the human blood fluke,Schistosoma mansoni

Abstract

The transposon piggyBac from the genome of the cabbage looper moth Trichoplusia ni has been observed in the laboratory to jump into the genomes of key model and pathogenic eukaryote organisms including mosquitoes, planarians, human and other mammalian cells, and the malaria parasite Plasmodium falciparum. Introduction of exogenous transposons into schistosomes has not been reported but transposon-mediated transgenesis of schistosomes might supersede current methods for functional genomics of this important human pathogen. In the present study we examined whether the piggyBac transposon could deliver reporter transgenes into the genome of Schistosoma mansoni parasites. A piggyBac donor plasmid modified to encode firefly luciferase under control of schistosome gene promoters was introduced along with 7-methylguanosine capped RNAs encoding piggyBac transposase into cultured schistosomula by square wave electroporation. The activity of the helper transposase mRNA was confirmed by Southern hybridization analysis of genomic DNA from the transformed schistosomes, and hybridization signals indicated that the piggyBac transposon had integrated into numerous sites within the parasite chromosomes. piggyBac integrations were recovered by retrotransposon-anchored PCR, revealing characteristic piggyBac TTAA footprints in the vicinity of the endogenous schistosome retrotransposons Boudicca, SR1, and SR2. This is the first report of chromosomal integration of a transgene and somatic transgenesis of this important human pathogen, in this instance accomplished by mobilization of the piggyBac transposon.

Atypical Mitogen-Activated Protein Kinase Phosphatase Implicated in Regulating Transition from Pre-S-Phase Asexual Intraerythrocytic Development of Plasmodium falciparum

“…Attenuated growth of the C9 MKP-null mutant remained stable over multiple subsequent generations, suggesting that survival was not due to phenotype reversion. This is consistent with general experience using the piggyBac system as it is now extensively used in a number of organisms, and the transposable elements remain integrated in the genome in the absence of transposase (31,(54)(55)(56)(57)(58). However, the extended maintenance of P. falciparum intraerythrocytic cultures required for transfection and the selection process in the experimental studies increases the possibility for secondary mutations to alter the cell cycle or cause growth attenuation.…”

Helminth infections: the great neglected tropical diseases

“…Similar findings have been described in schistosomes, and stage- and tissue-specific expression of schistosome genes has been investigated using reporter transgenes driven by the promoters of schistosome genes, including cathepsin L (125). Recently, Brindley and coworkers have described somatic transgenesis of S. mansoni facilitated by either the piggyBac transposon (126) or the murine leukemia retrovirus (127), including the first demonstration of integration of reporter genes into the chromosomes of a parasitic worm.…”

Vaccinomics for the Major Blood Feeding Helminths of Humans

“…Both vector-based RNAi and expression of foreign transgenes have begun to be deployed in studies with schistosomes, with potential outcomes including validation of putative vaccine candidates and even the overexpression of candidate antigens. Pseudotyped murine leukemia virus and the piggyBac transposon have both been shown to transduce developmental stages of schistosomes, leading to chromosomal integration of retroviral transgenes and transgene reporter activity Morales et al, 2007;Yang et al, 2010). Moreover, a vector-based RNAi approach has been reported in which the MLV transgene encoded a long hairpin RNA specific for a schistosome protease involved in hemoglobinolysis (Tchoubrieva et al 2010).…”