Developing low-background inducible expression technology for Leishmania donovani

开发杜氏利什曼原虫低背景诱导表达技术

• 批准号: 9045559
• 负责人: PHILLIP A YATES
• 金额: $ 19.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9045559
• 关键词: Address; Adopted; Biological Assay; Biology; Cell Separation; Characteristics; Coculture Techniques; Communities; Complex; Cutaneous; DNA; Development; Disease; Dissection; Endemic Diseases; Equilibrium; Exhibits; Fluorescence; Fluorescence-Activated Cell Sorting; Future; Genes; Goals; Green Fluorescent Proteins; Growth; Health; Human; Incidence; Knowledge; Knowledge acquisition; Laboratories; Leishmania; Leishmania donovani; Leishmaniasis; Luciferases; Metabolic; Molecular Genetics; Molecular Profiling; Parasite resistance; Parasites; Pathway interactions; Patients; Performance; Positioning Attribute; Poverty; Production; Property; Proteins; Publishing; RNA Interference; Reagent; Regulation; Renilla Luciferases; Reporter; Repression; Research; Series; Site; Staging; System; T7 RNA polymerase; Technology; Tetanus Helper Peptide; Tetracyclines; Transcript; Trypanosoma brucei brucei; Virulence; Visceral; Visceral Leishmaniasis; base; design; design and construction; differential expression; expression vector; genetic analysis; inducible gene expression; innovation; killings; knockout gene; meetings; new technology; new therapeutic target; novel; novel strategies; overexpression; pathogen; prevent; promoter; rapid technique; tool; transgene expression; vector

 DESCRIPTION (provided by applicant): The goal of this proposal is to develop a low-background inducible expression system for Leishmania donovani that will serve as a valuable tool for molecular genetic dissection of basic metabolic, regulatory and virulence pathways in this important human pathogen. L. donovani is the causative agent of visceral leishmaniasis, a devastating disease that is estimated to infect 500,000 people annually, and killing approximately 50,000 people per year. Current anti-leishmanial therapies can be costly, are often poorly tolerated and the incidence of resistant parasites is increasing. Rational design of new chemotherapeutic strategies critically depends the development and implementation of new technologies to accelerate the rate at which knowledge of the basic biology of this parasite is acquired. Inducible expression systems have been invaluable tools in the related kinetoplastid parasite T. brucei and the absence a suitable system in Leishmania has been a widely acknowledged deficiency in the field. Several inducible promoter systems have been described for Leishmania, yet none has been widely adopted by the Leishmania research community, in part due to high background expression in the uninduced state, poor inducibilty, or non-physiological expression levels. This proposal posits several parameters critical for optimal performance of inducible expression systems based on T7 RNA polymerase (T7 pol) and Tet repressor (TetR) in Leishmania. The three most important of these include, 1) preventing non-specific background expression; 2) attaining the proper balance of T7 pol and TetR co-expression; and 3) avoiding position effects conferred by the site of inducible expression construct integration. These and other parameters are addressed in two specific aims. The first specific aim focuses on the construction the T7 pol and TetR expression vectors, inducible reporter constructs and other DNA reagents that will constitute the tetracycline inducible T7 promoter expression system. This aim includes novel strategies to prevent background expression, to optimize the relative amounts and ratios of co-expressed T7 pol and TetR, and for streamlined vector construction. The goal of the second specific aim is to generate L. donovani lines encoding a tetracycline inducible expression system with low background and optimal induction properties. Parasite cultures co-expressing T7 pol and TetR at several different ratios, and encoding a tetracycline inducible, T7 promoter-driven Renilla luciferase-green fluorescent protein (Rluc-GFP) reporter construct will be subjected to consecutive rounds of fluorescence activated cell sorting to select parasites with no background (GFP negative) in the uninduced state, and high expression upon induction (GFP positive). To avoid position effects in clones shown by luciferase assays to have ideal characteristics, the Rluc-GFP reporter construct will be replaced with a construct that tags the locus for the integration of future inducible expression constructs.

 描述（由申请人提供）：本提案的目标是开发杜氏利什曼原虫的低背景诱导表达系统，该系统将作为对这种重要的人类病原体的基本代谢、调节和毒力途径进行分子遗传学剖析的有价值的工具。 L. donovani 是内脏利什曼病的病原体，这是一种毁灭性的疾病，估计每年感染 500,000 人，并导致约 100,000 人死亡每年有 50,000 人接受抗利什曼病治疗，费用昂贵，耐受性往往较差，而且新化疗策略的合理设计关键取决于新技术的开发和实施，以加快对利什曼病的认识。诱导表达系统是相关动质体寄生虫 T. brucei 的宝贵工具，而利什曼原虫缺乏合适的系统已被广泛认可。已经描述了利什曼原虫的几种诱导型启动子系统，但没有一个被利什曼原虫研究界广泛采用，部分原因是非诱导状态下的高背景表达、较差的诱导性或非生理表达水平。对于利什曼原虫中基于 T7 RNA 聚合酶 (T7 pol) 和 Tet 阻遏物 (TetR) 的诱导表达系统的最佳性能至关重要的几个参数，其中最重要的三个参数包括：1)。防止非特异性背景表达；2) 实现 T7 pol 和 TetR 共表达的适当平衡；以及 3) 避免诱导表达构建体整合位点带来的位置效应。具体目标侧重于构建 T7 pol 和 TetR 表达载体、诱导型报告构建体和其他 DNA 试剂，以构成四环素诱导型 T7 启动子表达系统。该目标包括防止背景的新策略。第二个具体目标是生成编码具有低背景和最佳诱导的四环素诱导表达系统的多诺瓦尼乳杆菌品系。寄生虫培养物以几种不同的比例共表达 T7 pol 和 TetR，并编码四环素诱导型、T7 启动子驱动的海肾荧光素酶绿色荧光蛋白。 (Rluc-GFP) 报告构建体将进行连续几轮的荧光激活细胞分选，以选择在未诱导状态下无背景（GFP 阴性）的寄生虫，并在诱导后高表达（GFP 阳性），以避免所示克隆中的位置效应。通过荧光素酶测定以获得理想的特性，Rluc-GFP报告构建体将被标记基因座的构建体所取代，以用于未来诱导表达构建体的整合。