Schistosoma mansoni: germ-line transformation approaches and actin-promoter analysis

Abstract

Towards germ-line transformation miracidia were biolistically transformed with GFP reporter gene constructs and successfully reintroduced into the schistosome cycle. By PCR and confocal microscopy the presence and the expression of GFP were confirmed in cercariae or adults of the F(0) and F(1) generations. This indicated the presence of the constructs in the germ-line, although no evidence for genome integration was obtained. About 3kb of 5' upstream sequences of the actin gene SmAct1 were identified by in silico analyses, and different fragments up to 1.5kb subcloned for GFP-vector construction. A 445bp fragment was sufficient for transcription initiation in larvae or adults as confirmed by confocal microscopy. An actin gene characteristic assembly of TATA, CArG, and CAAT boxes has been identified, which seems to be 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.