Development of antisense technique for the malaria parasite

疟疾寄生虫的基因靶向由于难以获得基因操作而受到阻碍。建立目标基因位点被破坏的寄生虫系需要3-4个月。由于疟原虫不存在用dicer沉默的机制，sirna介导的方法无法应用。因此，在这个项目中，我承担了开发针对恶性疟原虫寄生虫的反义技术。我制作了一个表达反义的EBA-175转录本的构建体，建立了耐药的寄生虫系，并评估了EBA-175的蛋白表达。结果我发现EBA-175仍有表达，而且针对反义特异性引物的RT-PCR无法扩增反义转录物。代替反义，我在这个区域插入GFP，发现GFP在已建立的寄生虫中也不表达。这些数据表明，构建的方向可能有问题，因为我将药物盒基因和基因靶向盒在一个从头到尾的方向上对齐。为了克服这个问题，我重新设计了构建体，使GFP和药物盒以头对头的方向定位，并发现GFP成功地从这个构建体中表达出来。利用这个基本构念，我制作了一组针对EBA-175的反义构念。为了开发一种可以快速评估基因重要性的检测方法，我将药物盒基因替换为荧光素酶基因，以便通过减少的寄生虫数量来检测必需基因，这代表了荧光素酶的活性。然而，当必需分子amal为靶点时，没有观察到显著差异。因此，很明显，这个系统需要更高的灵敏度。

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.