Development of antisense technique for the malaria parasite

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Gene targeting of the malaria parasite is hampered by the difficulty to obtain gene manipulation. It takes 3-4 months to establish parasite line for which target gene locus is disrupted. Because the silencing mechanism with dicer does not exist in malaria parasites, siRNA-mediated method cannot be applied. Thus in this project, I undertook to develop antisense technique against Plasmodium falciparum parasites. I made a construct expressing antisense against EBA-175 transcripts, which is known not essential for the parasite, establish drug-resistant parasite line, and evaluated the protein expression of EBA-175. As a result I found EBA-175 was still expressed and moreover RT-PCR against antisense-specific primer failed to amplify antisense transcript. Instead of antisense, I inserted GFP in this region and found that GFP was also not expressed in the established parasite. This data suggest that the orientation of the construct likely problem, because I aligned drug cassette genes and gene targeting cassette in a head to tail orientation. To overcome, I re-designed construct so that GFP and drug cassette locate in a head to head orientation and found that GFP was successfully expressed from this construct. Using this basic construct I made a panel of antisense constructs against EBA-175. To develop an assay with which essentiality of the gene can be evaluated quickly, I replaced drug cassette gene to Luciferase gene so that essential gene was detected by the reduced parasite number, which represents Luciferase activity. However, there are no significant difference was observed when essential molecule amal was targeted. Thus it became clear that the more sensitivity is needed for this system.