Optimizing gene suppression in schistosomes using RNA interference.
Schistosomes are parasitic platyhelminths that constitute an important public health problem globally. Here we describe optimized protocols for effectively suppressing gene expression in the intra-mammalian life stages of Schistosoma mansoni using RNA interference (RNAi). RNAi is a mechanism by which gene-specific double stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. The gene encoding the cysteine protease cathepsin B (SmCB1 or Sm31) was targeted by exposing the parasites to dsRNA encoding part of the cathepsin B coding region. Suppression was measured using quantitative real time PCR. Electroporation as a mode of dsRNA delivery was substantially more efficient (100-1000-fold) than simply soaking the parasites in an equivalent dose. Soaking the parasites with dsRNA in the presence of different proprietary liposome preparations did not enhance gene suppression. In fact, all three reagents tested were variably toxic to the cultured schistosomes. Both long dsRNAs as well as synthetic short inhibitory RNAs (siRNAs) were effective at eliciting gene suppression. Different siRNAs exhibited variable efficiencies of suppression, perhaps reflecting differences in siRNA accessibility to the cathepsin B mRNA. Parasites cultured in vitro for 7 days or more following their emergence from the intermediate snail host were more susceptible to RNAi than those treated with dsRNA on the day of emergence (during the process of cercarial transformation into schistosomula). In addition, adult male and female parasites (49 days old) were susceptible to RNAi. Using fluorescein-labeled dsRNA to monitor the process, it was seen that in schistosomula (cultured for 7 days), electroporated dsRNA entered primarily through the mouth into the caecum while in young parasites (freshly emerged from snails) dsRNA appeared to enter primarily into the pre- and post-acetabular glands. The cathepsin B gene was significantly suppressed for up to 40 days after treatment suggesting that, as in some other organisms, the RNAi process can be amplified in schistosomes.
Krautz-Peterson
Greice
G
Molecular Helminthology Laboratory, Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University, Cummings School of Veterinary Medicine, Grafton, MA 01536, USA.
Radwanska
Magdalena
M
Ndegwa
David
D
Shoemaker
Charles B
CB
Skelly
Patrick J
PJ
D000818:Animals; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D005821:Genetic Techniques; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism
D015801:Helminth Proteins; D012330:RNA, Double-Stranded; D002401:Cathepsin B