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Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A

of worms remained constant, and they recovered within a short period (Figure 2). PCR and RT-PCR analyses confirmed the presence and transcription of the transgene (results not shown). Western blot analysis showed that translation of the GFP occurred only in worms that had been stressed by heat shock, indicating that the regulatory elements worked as expected (Figure 3).

Bright-field microscopy demonstrated that pressures of 1,550 and 1,800 psi worked best, resulting in a high number and an equal distribution of microprojectiles in the worms (Figure 4A). Confocal laser scanning microscopy revealed green fluorescing signals occurring at the dorsal tegument (Figure 4B) as well as in the anterior and posterior portions of the worms (Figure 4C). Bombarded schistosomes that were not stressed by heat shock did not show any fluorescence (results not shown).

To determine whether the PDS-1000/He system is also suitable for transformation of larval schistosomes, we performed experiments with sporocysts generated in vitro. Here, lower pressures (900 and 1,350 psi) were applied. According to the constitutive expression of HSP70 in larval stages (Neumann et al. 1993), sporocysts were not heat shocked after bombardment. In a first series of experiments, up to 10,000 sporocysts were bombarded. The expression of GFP was verified by RT-PCR (results not shown) and immunoblotting (Figure 3). Confocal laser scanning microscopy revealed GFP expression within the larvae after biolistic treatment at 900 psi (Figure 5) as well as 1,350 psi. In control sporocysts (not bombarded), no fluorescence was detected (results not shown).


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