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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：该提案的目的是为Leishmania Donovani开发低背景的诱导表达系统，该系统将作为这种重要人类病原体中基本代谢，调节和病毒途径的分子遗传解剖的有价值工具。多诺瓦尼（L. donovani）是内脏利什曼病的病因，这是一种毁灭性的疾病，估计每年感染50万人，每年大约有50,000人丧生。当前的抗脊髓疗法可能会很昂贵，耐受性较差，并且抗性寄生虫的事件正在增加。新的化学治疗策略的合理设计在很大程度上取决于开发和实施新技术，以加速获得该寄生虫基本生物学知识的速度。诱导表达系统已成为相关的动型寄生虫T. brucei中的宝贵工具，而利什曼尼亚（Leishmania）的缺席在该领域已被广泛承认。利什曼尼亚（Leishmania）已经描述了几种诱导启动子系统，但利什曼尼亚研究界（Leishmania Research Crounders）尚未广泛采用，部分原因是在未诱导的状态下，较差的诱导性或非生理表达水平的背景表达很高。该提案提出的几个参数对于利什曼尼亚（Leishmania）中基于T7 RNA聚合酶（T7 POL）和TET抑制剂（TETR）的最佳性能至关重要。其中最重要的三个包括：1）防止非特异性背景表达； 2）实现T7 POL和TERTO共表达的适当平衡； 3）避免诱导表达构建体积分所赋予的位置效应。这些参数和其他参数以两个具体目的解决。第一个特定的目的侧重于构造T7 POL和TERTE表达载体，诱导的报告基因构建体以及其他将构成四环素诱导的T7启动子表达系统的DNA试剂。该目标包括防止背景表达的新策略，以优化共表达的T7 POL和TERT的相对量和比率以及简化的矢量构造。第二个特定目的的目的是生成编码四环素诱导的表达系统具有低背景和最佳诱导特性的L. donovani线。寄生虫培养物以几个不同的比率共表达T7 POR和TER，并编码诱导的四环素T7启动子驱动子驱动的肾luciferase荧光素酶绿色荧光酶 - 荧光蛋白（RLUC-GFP）记者构建体构建体将受到荧光分类的状态和高度（GFP）（GFP）的含量（gfp），将受到液体的状态（gfp）的影响（GFP）。积极的）。为了避免荧光素酶Assas显示具有理想特征的克隆中的位置效应，RLUC-GFP报告基因构建体将被替换为一个标记为整合未来诱导表达构建体的轨迹的构造。