New molecular techniques for T. cruzi

描述（由申请人提供）：克氏锥虫（T. cruzi）引起恰加斯病（例如，美洲锥虫）。这种感染是拉丁美洲的地方病;然而，由于来自流行地区的移民，在欧洲和美国都发现了恰加斯病。感染T. cruzi是发展为慢性感染，在急性感染后数十年出现疾病症状。对T. cruzi受到基因操作困难的限制。我们使用具有配体控制的去稳定化结构域（ddFKBP）的修饰的pTREX载体来调节感兴趣的基因/蛋白质。该载体系统允许快速和可逆的蛋白表达和不同T. cruzi生命周期阶段利用这种技术，我们发现两个丝裂原活化蛋白激酶（MAPK），TcMAPK 1和TcMAPK 3是T。克鲁兹我们计划在我们的ddFKBPpTREX载体系统的基础上开发一个条件性基因缺失系统。此外，在ddFKBP系统中以调节方式快速过表达致死基因应该是可行的，并且这可以在多个T. Cruzi分离株使用相同的载体构建体，而不需要对分离株进行任何遗传修饰。这种诱导致死表型T. Cruzi将非常适用于发病机理研究，其允许在感染后的不同时间点消除生物体以剖析疾病病因的机制。这些寄生虫也将促进免疫刺激的研究，并为开发针对这种感染的新疫苗策略提供数据。因此，我们提出：（1）利用T. cruzi增长，基于我们的ddFKBPpTREX载体。该系统可用于操纵该寄生虫的其他必需基因;（2）我们还将建立T。cruzi，允许毒素基因的调节表达，当这些基因表达时，这些基因将杀死这种寄生虫。

DESCRIPTION (provided by applicant): Trypanosoma cruzi (T. cruzi) causes Chagas Disease (e.g., American Trypanosomiasis) in humans. This infection is endemic to Latin America; however, due to immigration from endemic areas, Chagas Disease is found in both Europe and the United States. What is striking about infection with T. cruzi is the development of chronic infection with disease symptoms manifesting decades after the acute infection. Research on T. cruzi has been limited by the difficulties in genetic manipulation. We used a modified pTREX vector with a ligand-controlled destabilization domain (ddFKBP) to regulate a gene/protein of interest. This vector system allows rapid and reversible protein expression and efficient functional analysis of proteins in different T. cruzi life cycle stages. Using this technique, we found that two mitogen activated protein kinases (MAPK), TcMAPK1 and TcMAPK3, are essential for T. cruzi. We plan to develop a conditional gene deletion system based on our ddFKBPpTREX vector system. In addition, quickly over-expressing a lethal gene in a regulated fashion should be feasible in the ddFKBP system and this can be done in multiple T. cruzi isolates using the same vector construct without any need to genetically modify the isolates. Such an inducible lethal phenotype T. cruzi would be very useful for pathogenesis studies allowing elimination of the organism at various time points after infection to dissect the mechanisms of disease causation. These parasites would also facilitate studies on immune stimulation and provide data for the development of new vaccine strategies for this infection. We, therefore, propose to: (1) develop a robust conditional knockout vector system using TcMAPK1 and TcMAPK3, essential genes for T. cruzi growth, based on our ddFKBPpTREX vectors. This system should be useful for the manipulation of other essential genes in this parasite; and (2) we will also create vector systems for T. cruzi that allow the regulated expression of toxin genes that will kill this parasite when these genes are expressed.