Heterogenous Expression Of Enhanced Green Flurescent Protein (EGFP) Gene In Schistosoma Japonicum And In Vitro Primary Cultured Cells Of Schistosomula

Objective: To explore the possibility of heterogenous gene expression in primary cultured cells from schistosomula as well as in juvenile and adult S. japonicum and application of electroporation in transformation of schistosomula and adults for laying a foundation for the further study of transgenic schistosoma.Methods: After preparation of primary cultured cells from Schistosomula, the EGFP genes were introduced into the cells with lipofectamine and the expression was observed by fluorescent microscopy. The plasmids of pEGFP-Cl were introduced into mechanically transformed Schistosomula and aduls with electroporation. The presence, transcription and translation of transgene in electroporated Schsitosomula and aduls were confirmed by PCR, RT-PCR and Western blot analysis, respectively using the genomic DNA, total RNA and protein extracted and isolated from them. Localization of EGFP within electroporated schistosomula and adults was performed with confocal laser scanning microscopy. Finally, the electroporated schistosomula were assessed for infectivity in mice by intramuscular injection. Results: Fluorescent primary cultured cells from Schistosomula were identified. 760 bp and 276 bp amlified products by PCR and RT-PCR were found coincide with the expected size and expression of EGFP gene in electroporated Schistosomula and adults was confirmed by western blotting. Fluorescence of EGFP was localized in tegument and subtegument of the electroporated...