mansoni HSP70 promoter and terminator. This plasmid was introduced into sporocysts and adults, and expression of GFP could be shown after heat shock induction by confocal microscopy 3 days after transfection. Fluorescence was mainly visible on the surface of adult worms and inside sporocysts. The authors also employed RT-PCR to detect GFP transcripts and Western blotting to identify the GFP protein (13). Expanding on this work, Wippersteg and Romidepsin co-workers (15) then exchanged the SmHSP70 promoter and terminator

elements in the plasmid with cis-acting elements of the S. mansoni ER60 (SmER60) gene. SmER60 encodes a cysteine protease which in earlier studies had been localized to the endoplasmic reticulum in excretory tissues in adult parasites (17). After bombardment of sporocysts, the expression of GFP was observed to be tissue-specific, and the localization of ER60 in the excretory/secretory (ES) system of the larval parasites suggested that ER60 might have a role in penetration and migration of miracidia in the intermediate snail host. In an additional follow-up report, the same authors verified this tissue-specific expression of the ER60 protease by employing Texas Red-labelled BSA, which accumulates in the ES system,

together with biolistic transformation. The ER60-GFP and the Texas Red-BSA co-localized in the same compartments (16). The same approach to co-localize Texas Red-BSA to the ES system was used by Rossi et al. (14) to study S. mansoni BTK pathway inhibitors calcineurin A and its expression in the ES system. Similar to the results discussed previously, fluorescent signals for GFP and Texas Red co-localized in the ES system of the parasite. We introduced plasmid DNA by particle bombardment into miracidia, sporocysts and adults of S. mansoni (12). We particularly focused on the miracidial life cycle stage, because this larval stage offers the unique opportunity to introduce transgenes into the

germline and additionally to reintroduce transgenic organisms into the parasite life cycle. Bombarded miracidia were able to infect Biomphalaria ifenprodil glabrata snails, and particles could clearly be identified within the developing sporocysts in paraffin sections of infected snail tissues. Interestingly, these gold particles were located close to the nuclei of germ ball cells, suggesting germline transfection and the derivation of transgenic schistosomes is feasible. RT-PCR showed that the reporter gene was still transcribed 10 days after infection of the snails with transgenic miracidia. These findings indicated that it is possible to return transfected miracidia to the parasite life cycle, a crucial step for the establishment of stable transgenesis in schistosomes. A similar approach was taken by Beckmann et al. (18). Miracidia were biolistically transformed with GFP reporter gene constructs and reintroduced into the life cycle.