Transgene expression in Schistosoma mansoni: introduction of RNA into schistosomula by electroporation.
Despite their significance in human and veterinary medicine, and the ability to maintain the parasites in the mouse, relatively little functional detail is available regarding the biology of schistosomes. This deficit is due largely to the lack of well-developed molecular tools for manipulating gene expression in these parasites. Here, we describe an electroporation protocol that provides a routine approach for efficiently introducing nucleic acids into schistosomes. Using luciferase-encoding RNA for electroporation, and luciferase activity as a read-out, we established 400 microg/ml of RNA, and a 20 ms pulse at 125 V using a square wave electroporation generator to be optimal for electroporating schistosomes. Under these conditions schistosomula from 1 hr to 18 hr old could be successfully electroporated, the majority of parasites within a population expressed the introduced RNA, and acute mortality was negligible. Electroporation, as described here, makes possible experimental studies using transiently expressed constitutively active and/or dominant negative mutant proteins, etc. In addition, the finding that electroporation can be used to introduce RNA into schistosomula raises the possibility of using this approach to introduce either DNA constructs or dsRNA sequences, both of which might be expected to have longer-term, ideally inheritable, effects.
Correnti
Jason M
JM
Department of Pathobiology, University of Pennsylvania, 203D Johnson Pavilion, 3610 Hamilton Walk, Philadelphia 19104-6076, USA.
Pearce
Edward J
EJ
D000818:Animals; D018274:Electroporation; D015870:Gene Expression; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012550:Schistosoma mansoni / Q000235:genetics*; D019076:Transgenes
D012313:RNA; D008156:Luciferases