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Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A

penetrate cell layers of the target and introduce genetic material into living cells. The microprojectiles we used were gold particles, which are uniform in size and do not catalytically attack DNA. In addition, gold is biologically inert and is not toxic to most cells (Sanford et al. 1993).


Methods
Transformation Vector

Figure 1 shows the transformation vector used in this study. The vector contained the inducible regulatory elements of the heat shock protein 70 (HSP70) gene from S. mansoni and the Green Fluorescent Protein (GFP) gene as the reporter gene.

Parasite Stock and Culture Conditions
A Liberian strain of S. mansoni was maintained in snails, Biomphalaria glabrata, and in Syrian hamsters, Mesocricetus auratus. Adult worms were obtained by perfusion at day 42 post-infection, and washed 3 times with RPMI 1640 containing 10% fetal calf serum, before they were kept in 60 mm petri dishes at 37C, 5% CO2 (Grevelding et al. 1997). Isolation of miracidia and in vitro transformation to sporocysts were done as described elsewhere (Wippersteg et al. 2002).

Coating of Microparticles
Gold particles (0.61.6 m) were coated with vector DNA purified by chromatography on a Macherey-Nagel column. For experiments using the PDS-1000/He system, microprojectiles for five bombardments were prepared by mixing 50 l of gold (in 60 mg/ml 50% glycerol), 525 g of DNA (0.21 g/l), 40 l of spermidine (0.05 M, free base), and 50 l of CaCl2 (2.5 M). After 3 min of vortexing, the gold particles were pelleted by centrifugation, and the supernatant was removed. The pellet was washed 2 times with ethanol (100%) by pipetting up
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