piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni.
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.
Morales
Maria E
ME
Department of Tropical Medicine, Tulane University, Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112, USA.
Mann
Victoria H
VH
Kines
Kristine J
KJ
Gobert
Geoffrey N
GN
Fraser
Malcolm J
MJ
Kalinna
Bernd H
BH
Correnti
Jason M
JM
Pearce
Edward J
EJ
Brindley
Paul J
PJ
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004251:DNA Transposable Elements; D018274:Electroporation; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012550:Schistosoma mansoni / Q000235:genetics*; D019076:Transgenes
D017931:DNA Primers; D004251:DNA Transposable Elements