A Resource Center for Tetrahymena Thermophila

嗜热四膜虫资源中心

• 批准号: 10591414
• 负责人: THEODORE G CLARK
• 金额: $ 12.39万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591414
• 关键词: Applied Research; Autonomous Replication; Biological; Biology; CRISPR/Cas technology; Catalytic RNA; Cell Line; Cell Nucleus; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Country; Development; Disease; Engineering; Gene Rearrangement; Genome; Goals; Guide RNA; Health; Human; Investigation; Macronucleus; Maintenance; Mediating; Methodology; Microbe; Modification; Mutagenesis; Mutation; Nuclear; Organism; Partner in relationship; Pharmaceutical Preparations; Production; Proteins; Research; Research Personnel; Resources; Ribosomal DNA; Scientist; Services; System; Telomerase; Testing; Tetrahymena; Tetrahymena thermophila; Transgenes; dimorphism; expression vector; genome editing; human disease; human model; improved; innovation; insight; micronucleus; mutant; new technology; next generation; protein expression; rRNA Genes; tool; uptake; vector

Project Summary / Abstract Ciliates are unicellular yet possess much of the cellular complexity found in multicellular organisms. They have evolved substantial biological innovations (e.g. nuclear dimorphism, programmed DNA rearrangements), the studies of which have revealed fundamental insights into living systems. Even so, they remain an understudied class of microbes. Tetrahymena in particular has had huge impact on biology, including the discovery of ribozymes and telomerase, and continues to contribute to our understanding of basic mechanisms critical to human health and disease. The Tetrahymena Stock Center is the primary resource for the maintenance and distribution of genetically defined strains of Tetrahymena thermophila to researchers in this country and abroad. This project aims to develop core new technologies to manipulate the genome of Tetrahymena and thereby enable researchers to more adeptly contribute to the basic understanding of cellular processes. A major goal of the project is to adapt CRISPR-Cas9-mediated genome editing for use in Tetrahymena, specifically to promote more efficient modification of the germline genome. Targeting of Cas9 and guide RNAs specifically to the micronucleus will lead to specific alterations of this silent germline nucleus rather than that of the expressed somatic nucleus. Specific modification of the silent germline will allow Tetrahymena researchers to create and subsequently study deleterious mutations when they are brought into the expressed nucleus in the next generation. A major goal for this applied research is to lower barriers that impede the use of Tetrahymena to study fundamental biology and model human disease. Tetrahymena also offers many advantages as a protein expression system. To improve the stability of high level expression from ribosomal DNA-based expression vectors, the project aims create cell lines deleted for the single-copy rRNA gene in the germline and thereby eliminate competition between the endogenous rDNA and introduced expression vectors, which will provide for more reliable protein production. Development of these approaches will enable mutagenesis and vector-based transformation without the need for drug selection. Successful attainment of these goals will assist numerous scientist is their investigations. These methodologies and associated resources will be distributed through the Stock Center and will be used to expand the services offered by the Center to the research community with an additional goal of increasing the Center’s sustainability.

项目总结/摘要 纤毛虫是单细胞的，但具有多细胞生物中发现的大部分细胞复杂性。他们有 进化出实质性的生物学创新（例如核二型性，程序化DNA重排）， 对其的研究揭示了对生命系统的基本见解。即便如此，他们仍然是一个替补 一类微生物。特别是四膜虫对生物学产生了巨大的影响，包括发现 核酶和端粒酶，并继续有助于我们理解的基本机制， 人类健康和疾病。四膜虫库存中心是主要的资源， 向国内外的研究人员分发了嗜热四膜虫的遗传定义菌株。 该项目旨在开发核心新技术来操纵四膜虫的基因组，从而 使研究人员能够更熟练地为细胞过程的基本理解做出贡献。的一个主要目标 该项目是调整CRISPR-Cas9介导基因组编辑以用于四膜虫，特别是促进 更有效地修饰生殖细胞基因组。Cas9和向导RNA特异性靶向 微核将导致这种沉默的生殖细胞核的特异性改变，而不是表达的 体细胞核沉默种系的特定修饰将使四膜虫研究人员能够创造和 随后研究有害的突变，当它们被带入下一个表达的细胞核时， 一代这项应用研究的一个主要目标是降低阻碍四膜虫利用的障碍， 研究基础生物学和人类疾病模型。四膜虫作为一种蛋白质也有许多优点 表达系统提高核糖体DNA表达高水平表达的稳定性 载体，该项目的目的是建立细胞系删除的单拷贝rRNA基因在种系，从而 消除内源rDNA和引入的表达载体之间的竞争，这将提供 更可靠的蛋白质生产。这些方法的发展将使诱变和基于载体的 不需要药物选择就能转化。成功实现这些目标将有助于许多 科学家是他们的研究。这些方法和相关资源将通过 股票中心，并将用于扩大该中心提供的服务，以研究界与 增加中心的可持续性的另一个目标。