These results suggested that the construct might have been submitted through the germline although no proof for genome integration was obtained. Taken together, PF-01367338 concentration the articles by Heyers et al. and Beckmann et al. (12,18) show proof of principle that it might be possible to enter the germline using transformed miracidia. A further publication by Wippersteg et al. (19) reports the tissue-specific
expression of GFP driven by the promoters of two S. mansoni protease genes cathepsin L1 and cathepsin B2. As predicted from earlier reports (20), the S. mansoni cathepsin L1 promoter drove GFP expression throughout the gut whereas transformation with the SmCB2 (21) construct resulted in GFP fluorescence localized in the tegument. Particle bombardment was also employed by Beckmann et al. (18). Here, different reporter gene constructs using the S. mansoni actin1 regulatory elements and GFP as reporter KU57788 gene were used for transient transformation of adult males and sporocysts. A 445-bp promoter fragment was sufficient for transcription initiation in larvae or adults as confirmed by confocal microscopy. Actin gene characteristic TATA, CArG and CAAT boxes were identified in the promoter, suggesting that it is functionally conserved between vertebrates and invertebrates. However, a vertebrate-specific intron containing an additional regulatory CArG box was not found indicating that
the regulation of SmAct1 transcription depends exclusively on its promoter region. In addition, the authors showed GFP expression in the tegumental area, especially the tubercles, in the muscle tissue
and weakly in the parenchyma of the male worms. The most recent publication describing the transfection of schistosomes second using biolistic methods was only published last year (22). Here, modified reporter gene constructs containing 5′ and 3′ regulatory regions of protease genes (cathepsins F and D) were used to transfect immature adult worms. The results obtained showed that there was a minor improvement of the intensity and distribution of the reporter signal in constructs containing parts of the ORF and/or 3′ gene-specific genomic fragments. However, reporter signals were found in tissues other than the gut and the authors suggest that this might represent dysregulated transcription which could impact on the utility of biolistics as a tool to accurately profile spatial expression of transgenes. Electroporation as a tool to introduce plasmid-based DNA constructs was tested in S. japonicum and S. mansoni (23,24). Yuan et al., using a commercial plasmid (pEGFP-C1), showed that the cytomegalovirus (CMV) promoter was able to drive EGFP expression in primary cell cultures of S. japonicum. Introduction of the plasmid into schistosomula and adult worms by electroporation led to EGFP expression as demonstrated by RT-PCR, Western blotting and confocal microscopy with EGFP fluorescence detectable along the tegumental surface of the worms (24).