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Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A

ding to standard protocols.

Microscopic analyses of bombarded worms were done 24 hr after heat shock. Adults were individually placed onto glass slides and carefully fixed with cover slips. Larval stages were analyzed by microscopy 48 hr after bombardment. Microscopy was performed with a Leica TCS NT confocal laser scanning microscope using the GFP-defined wavelength of 488 nm for excitation. Laser activity was adjusted to 5060%.


Results
To investigate whether biolistic gene transfer can be used to generate transgenic schistosomes, bombardment experiments were performed with two different systems, the Helios gene gun and the PDS-1000/He. In both cases the hsp70-GFP vector was used for transformation.

With the gene gun, a total of 328 worms were bombarded in seven independent experiments. Due to air pressure conditions, nearly 1/3 of the worms were lost during bombardment. The remaining schistosomes were kept under standard culture conditions until heat shock to induce reporter gene activity. After this treatment, the survival rate decreased below 50%. Light microscopy showed gold particles within the remaining worms, and also on the tegument. The presence and transcription of the transgene in bombarded worms were confirmed by PCR/RT-PCR with extracted DNA/RNA and GFPspecific primers. Fluorescence microscopy showed green areas in the worms indicating GFP activity (results not shown).

In contrast to the air-pressure conditions of gene gun treatment, the PDS-1000/He system works with a vacuum chamber. After bombardment, the worms were still present in the middle of the petri dish, and nearly all worms survived the procedure. Even after heat shock, the number
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