A Resource Center for Tetrahymena Thermophila

嗜热四膜虫资源中心

• 批准号: 10380159
• 负责人: THEODORE G CLARK
• 金额: $ 12.72万
• 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/10380159
• 关键词: Applied Research; Biological; Biology; CRISPR/Cas technology; Catalytic RNA; Cell Line; Cell Nucleus; Cell physiology; Cells; Chromosomes; 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; base; 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.

项目摘要 /摘要 纤毛是单细胞的单细胞，但潜在的许多细胞复杂性在多细胞生物中发现。他们有 进化的实质生物学创新（例如核二态，编程的DNA重排）， 其中的研究揭示了对生活系统的基本见解。即使这样，他们仍然是一种理解 类微生物类。尤其是四膜虫对生物学产生巨大影响，包括发现 核酶和端粒酶，并继续有助于我们对基本机制至关重要的理解 人类健康和疾病。四捕虫储备中心是维护和维护的主要资源 一般定义的四膜菌嗜热菌株的分布给该国和国外的研究人员。 该项目旨在开发核心新技术来操纵四膜虫的基因组，从而 使研究人员能够更适当地有助于对细胞过程的基本理解。一个主要目标 该项目是为了适应用于四氢菌的CRISPR-CAS9介导的基因组编辑，特别是为了促进 种系基因组的更有效修饰。针对CAS9和指导RNA专门针对 微核将导致这种无声种系核的特定改变，而不是表达的核 体核。静默种系的特定修改将使四膜研究人员能够创建和 随后研究在下一个将它们带入表达的核中时删除了突变 一代。这项应用研究的一个主要目标是降低阻碍使用四氢菌的障碍 研究基本生物学和模型人类疾病。四膜虫还具有许多优势作为蛋白质 表达系统。提高核糖体DNA表达高水平表达的稳定性 向量，该项目的目的是创建针对种系中单拷贝rRNA基因删除的细胞系，从而 消除内源性rDNA和引入表达向量之间的竞争，这将提供 更可靠的蛋白质产生。这些方法的开发将使诱变和基于向量的诱变 不需要药物选择的转化。这些目标的成功尝试将有助于许多 科学家是他们的调查。这些方法和相关资源将通过 股票中心，将用于扩展该中心提供的服务给研究社区 提高中心可持续性的其他目标。