Figure 5.

Gene suppression of S. mansoni cathepsin D (SmCD) in schistosomules. Seven, nine and 11day old schistosomes were transformed by electroporation with 30 μg of SmCD 0.5 kb-dsRNA or Luc-dsRNA, and then cultured in vitro. Forty eight hours and seven days later, batches of schistosomules were harvested for analysis of (A) total RNA by RT-PCR and (B) aspartic protease activity. At 48 h following electroporation, knockdown of both SmCD transcripts and aspartic protease activity was apparent (not shown). By seven days, SmCD transcription was completely silenced (A) and protease activity profoundly suppressed (B). Panel A: RNA was 10-fold serially diluted for analysis by RT-PCR. The dilution series reflects input of 50.0, 5.0, 0.05 ng of total parasite RNAs. Two other genes, SmCB1 and GAPDH, were quantified by RT-PCR to monitor for possible off-target effects of dsRNA exposure. Panel B: SmCD protease activity was measured at pH 3.5 in soluble extracts of schistosomules using the cathepsin D-specific substrate MCA-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-Arg. Extracts of SmCD-dsRNA treated schistosomules exhibited markedly less protease activity than the Luc-dsRNA treated controls. For all three age groups of the schistosomules, significant suppression of SmCD was evident (P ≤ 0.05 in each case), although there was a trend for diminished silencing with increasing age of the schistosomules for seven to nine to 11 days of age.