New molecular techniques for T. cruzi

克氏锥虫的新分子技术

• 批准号: 8660617
• 负责人: Huan Huang
• 金额: $ 20.32万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-13 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8660617
• 关键词: Achalasia; Acute; Address; African Trypanosomiasis; Area; Biology; Cardiomyopathies; Cellular biology; Chagas Disease; Chronic; Clinical; Colon; Complication; Data; Development; Disease; Dominant-Negative Mutation; Employee Strikes; Esophagus; Essential Genes; Etiology; Europe; Gene Deletion; Gene Proteins; Genes; Genetic Techniques; Genome; Growth; Heart failure; Human; Immigration; Immune; Immune response; Infection; Kinetics; Knock-out; Latin America; Lethal Genes; Life Cycle Stages; Ligands; Methods; Mitogen-Activated Protein Kinases; Molecular; Molecular Genetics; N-terminal; Organ; Organism; Parasites; Pathogenesis; Patients; Pharmaceutical Preparations; Phenotype; Play; Polymerase; Protein Analysis; RNA Interference; Regulation; Research; Role; Symptoms; System; Techniques; Tetracyclines; Time; Toxin; Transfection; Transgenic Organisms; Trypanosoma brucei brucei; Trypanosoma cruzi; United States; Vaccine Design; Vaccines; Visceral; Work; base; design; genetic manipulation; improved; insight; interest; killings; knockout gene; mortality; novel strategies; novel vaccines; pathogen; protein expression; public health relevance; vector

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.

描述（由申请人提供）：Cruzi锥虫（T. Cruzi）引起人类的Chagas病（例如，美国锥虫病）。这种感染是拉丁美洲特有的。但是，由于流行地区的移民，在欧洲和美国都发现了查加斯病。引人注目的克鲁齐（T. cruzi）感染是急性感染后数十年来表现出疾病症状的慢性感染的发展。关于克鲁兹的研究受到遗传操作困难的限制。我们使用了带有配体控制的不稳定结构域（DDFKBP）的改良PTREX载体来调节感兴趣的基因/蛋白质。该媒介系统允许在不同T. Cruzi生命周期阶段快速，可逆的蛋白质表达和有效的蛋白质功能分析。使用这种技术，我们发现两个有丝分裂原活化的蛋白激酶（MAPK），TCMAPK1和TCMAPK3对于T. Cruzi来说至关重要。我们计划基于我们的DDFKBPPTREX矢量系统开发条件基因缺失系统。此外，在DDFKBP系统中应以受调节方式快速表达致命基因，并且可以使用相同的矢量构建体在多个T. cruzi分离株中进行，而无需任何遗传修改分离株。这种可诱导的致命表型T. cruzi对于发病机理研究非常有用，允许在感染后的各个时间点消除生物体以剖析疾病因果的机制。这些寄生虫还将促进有关免疫刺激的研究，并为这种感染的新疫苗策略提供数据。因此，我们建议：（1）基于我们的ddfkbbpptrex载体，使用TCMAPK1和TCMAPK3（用于Cruzi生长的基本基因）开发出强大的条件敲除矢量系统。该系统对于在该寄生虫中对其他必要基因的操纵应该有用。 （2）我们还将为T. cruzi创建向量系统，以允许在表达这些基因时杀死该寄生虫的毒素基因的调节表达。