High-throughput Gene Knockout Production in Tetrahymena thermophila

描述（由申请人提供）：该项目的具体目标是产生含有约1300个嗜热四膜虫蛋白质编码基因基因替换突变的大肠杆菌克隆，并将这些克隆转化为四膜虫种系基因组以产生敲除菌株。克隆和菌株将被低温保存，并免费提供给任何感兴趣的研究人员。这一资源将极大地促进活跃的研究团体使用这种模式生物的努力，并吸引新的研究者进行研究。四膜虫已经在实验室中研究了近一个世纪，并在基础和生物医学研究中贡献了许多重要的发现。主要研究领域包括染色质修饰、RNA干扰、端粒形成和维持、细胞骨架动力学、分泌、DNA复制、扩增和消除。全基因组序列和注释信息的可用性为该模型开辟了新的研究领域，吸引了新的研究者。四膜虫保留了许多祖先真核生物的遗传功能，这些功能在其他单细胞模式生物（如酵母）中已经丢失。这些基因中有许多与疾病相关的人类同源基因。由于四膜虫只经历同源基因替换，许多这些基因已经通过创建敲除来研究。四膜虫对这种分析的独特优势是，必要基因的敲除可以在转录沉默的种系微核中维持，并通过简单的交配进入表达。然而，迄今为止，敲除构建体和菌株的产生是在低效的、逐个基因的基础上进行的，这阻碍了四膜虫研究的进展，也阻碍了对更大基因家族的研究。通过优化和集中这一过程，我们打算克服这一障碍，长期提高四膜虫研究界的能力。大量研究人员对这一高通量项目表达了热情，并确定了许多特别感兴趣的敲除靶点。敲除菌株将转移到四膜虫库存中心，供所有研究人员使用。所开发的方法将作为未来努力产生一整套四膜虫敲除菌株的模型。

DESCRIPTION (provided by applicant): The specific aims of this project are to generate E. coli clones containing gene replacement mutations in approximately 1,300 protein-coding genes of Tetrahymena thermophila and to transform these clones into the Tetrahymena germline genome to generate knockout strains. The clones and strains will be cryogenically preserved and made freely available to any interested researcher. This resource will greatly facilitate the efforts of an active research community using this model organism and attract new investigators to its study. Tetrahymena has been studied in the laboratory for nearly a century and has contributed to many discoveries of fundamental importance in basic and biomedical research. Prominent areas of investigation include chromatin modification, RNA interference, telomere formation and maintenance, cytoskeletal dynamics, secretion, and DNA replication, amplification and elimination. The availability of whole genome sequence and annotation information has opened up new fields of investigation and attracted new researchers to this model. Tetrahymena has retained many ancestral eukaryotic genetic functions that have been lost in other unicellular model organisms, such as yeast. Many of these genes have human orthologs that are disease- associated. Because Tetrahymena undergoes exclusively homologous gene replacement, many of these genes have been studied by creating knockouts. A unique advantage of Tetrahymena for such analyses is that knockouts of essential genes can be maintained in the transcriptionally silent germline micronucleus and brought into expression by a simple mating. However, the generation of knockout constructs and strains has thus far proceeded on an inefficient, gene-by-gene basis that has hindered the progress of Tetrahymena research and precluded investigation of larger gene families. By optimizing and centralizing this process, we intend to overcome this hurdle for the long-term enhancement of the capabilities of the Tetrahymena research community. A large number of researchers have expressed enthusiasm for this high throughput project and identified numerous knockout targets of particular interest. The knockout strains will be transferred to the Tetrahymena Stock Center and made available to all investigators. The methods developed will serve as a model for future efforts to generate a complete set of Tetrahymena knockout strains. PUBLIC HEALTH RELEVANCE: Research with the simple model organism Tetrahymena has contributed to our understanding of several fundamental processes, such as how chromosome ends replicate; that particular study advanced studies of aging and cancer. Numerous other basic studies with far-reaching relevance to human health would be facilitated by the availability of the proposed freely available resource.