HSP70-controlled GFP expression in transiently transformed schistosomes.
Among the parasitic helminths schistosomes are of high medical and economic importance. Despite of the world-wide relevance of this parasite, very little is known about the cellular mechanisms controlling its development and concerning the host-parasite interaction. Within the last decade a great effort has been made in this blood fluke to identify genes which play important roles during these processes. However, molecular analysis was limited by the fact, that neither function nor regulation of candidate genes could be investigated in this organism due to the lack of transformation protocols. Here, we present the strategy of ballistic gene transfer to introduce and characterize transgenes in different schistosome life stages. As a transformation vector, the heat shock protein 70 (hsp70) gene promoter and terminator from Schistosoma mansoni were cloned and fused to the green fluorescent protein (GFP) reporter gene. In a first attempt, the hsp70--GFP vector was successfully tested in a eukaryotic cell line. Thereafter, adult male schistosomes and sporocysts were transformed with this vector, and GFP expression was demonstrated using molecular and microscopical methods. PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses confirmed the presence, transcription and translation of the transgene in adults. Confocal laser scanning microscopy revealed GFP-activity at various sites along the surface of the worms after hs induction and within sporocysts. These results suggest diverse roles for hsp70 during the development of schistosomes. Furthermore, the results demonstrate the feasibility of this method and open the perspective to analyze a variety of molecular functions in schistosomes.
Wippersteg
Volker
V
Institute for Genetics, Genetic Parasitology and Center for Biological and Medical Research, Heinrich-Heine-University, Universitatsstrasse 1, D-40225, Dusseldorf, Germany.
Kapp
Katja
K
Kunz
Werner
W
Jackstadt
W Peter
WP
Zahner
Horst
H
Grevelding
Christoph G
CG
D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D012550:Schistosoma mansoni / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D019076:Transgenes
D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins