( and down and then concentrated by cen...)Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A

For experiments with the gene gun, DNA-coating and loading of gold projectiles into Tefzel tubing were done according to Jackstadt et al. (bulletin 2433).

Bombardment and Heat Shock Procedures
For gene delivery experiments with adult schistosomes, approximately 25 males were cultured at 37C in 60 mm petri dishes for 1 day after collecting them from the final host. Immediately before bombardment, the medium was carefully removed, and the worms were placed in the middle of the dish. Biolistics were performed using the following parameters: 1.3502.000 psi helium pressure, target distance stage 1 (3 cm), and 1520"Hg chamber vacuum. After bombardment, medium was added, and the worms were cultivated for an additional 24 hr. Heat shock was applied for 4 hr at 42C.

For biolistic experiments with sporocysts, groups of 2,000 4,000 individuals were pooled and transferred to 35 mm petri dishes. Pressures of 900 and 1,350 psi were applied at a distance of 3 cm and a vacuum of 1520"Hg. After bombardment, 2 ml of culture medium were added, and the larvae were cultured for an additional 48 hr without heat shock.

Molecular and Microscopic Analyses
DNA or RNA for PCR or RT-PCR analyses was isolated from bombarded worms that had been frozen in liquid nitrogen 6 hr after heat shock (Neumann et al. 1993). Protein from bombarded adults was extracted 24 hr after heat shock (Wippersteg et al. 2002). Protein from larval stages was extracted 48 hr after bombardment. SDS-PAGE and western blots were done accor
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