TFTag: A novel library of tagged transcription factors in Drosophila

TFTag：果蝇中标记转录因子的新型文库

• 批准号: BB/W018780/1
• 负责人: Korneel Hens
• 金额: $ 145.8万
• 依托单位: Oxford Brookes University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW018780%2F1
• 关键词: TFTag; novel; library; tagged; transcription

Every multicellular organism consists of dozens or even hundreds of different cell types that are defined by their different protein repertoires. The building plans for the proteins are encoded in the form of genes in the DNA, as are the instructions of where and when to make them. This process, referred to as gene expression, is highly dynamic and is constantly adjusted during development or in response to, e.g., environmental changes. Each cell type has its characteristic pattern of gene expression, and mis-regulation is associated with various diseases, including cancer. Therefore, gene expression must be tightly regulated to ensure that each cell has the correct protein content. This is largely done by a specific class of proteins, the transcription factors (TFs), which can bind to DNA and cooperate with each other to influence which genes are transcribed in what quantities. This in turn determines which proteins are present in which cells and in which amount. TFs are, accordingly, studied by a wide range of scientists in the life and medical sciences.Research on TFs mainly seeks to uncover information about their expression level, localisation, binding sites in the genome, interaction partners, and effects of a loss of function. Most of these analyses rely heavily on the use of antibodies, the production of which is costly, difficult, and ethically questionable. Moreover, methods involving antibodies are geared towards fixed biological materials, which makes the recapitulation of dynamic processes challenging. A common solution is the use of genetic tricks to express a version of the TF that is fused to another protein, which can, in turn, be readily detected and is, accordingly, called a tag. However, tags are often suitable for only a single or few methods or otherwise are large and might interfere with the function of the TF. Moreover, the additional expression of the tagged TF can induce artefacts. Also, tools for the expression of such tagged constructs are only available for a subset of TFs.The fruit fly, Drosophila melanogaster, has long served as one of the prime model organisms to study basic biological processes. It benefits from a large, well-connected worldwide community of researchers and the availability of vast genetic and molecular resources. Importantly, it allows investigations in vivo, and the creation of transgenic lines for the generation of novel tools is straightforward.Here we propose to create a biological and bioinformatic resource consisting of a library of transgenic fly lines in which all Drosophila TF genes have been tagged at their native genomic locus. We will also establish a database with basic data on the expression and DNA binding of a subset of TFs that have not previously been studied. Our resources will alleviate many of the shortcomings of previous libraries of tagged proteins: 1) Our tagging strategy allows for the easy exchange of the tag, making each TF accessible for various downstream applications; 2) We will create individual transgenic lines in which either end of a TF has been tagged, which reduces the risk of an interference of the tag with protein function; 3) For TFs that are expressed with alternative ends, we will create a line for each end, allowing the study of different versions of each TF. We will distribute the fly lines free of charge to members of the research community, and the database will, likewise, be free to use. These resources will open the possibility to study any Drosophila TF with the methods of choice in any biological process imaginable and allow scientists to easily identify potential candidates among the many unstudied TFs. We expect that our resource will greatly advance research in the field of gene regulation.

每个多细胞生物体都由数十种甚至数百种不同的细胞类型组成，这些细胞类型由其不同的蛋白质库定义。蛋白质的构建计划以基因的形式编码在DNA中，就像在哪里和何时制造它们的指令一样。这一过程被称为基因表达，是高度动态的，并且在发育期间或响应于，例如，环境变化。每种细胞类型都有其特有的基因表达模式，错误调节与各种疾病有关，包括癌症。因此，基因表达必须受到严格调控，以确保每个细胞具有正确的蛋白质含量。这主要是由一类特定的蛋白质，转录因子（TF）完成的，它们可以与DNA结合并相互合作，以影响哪些基因以何种数量转录。这反过来又决定了哪些蛋白质存在于哪些细胞中以及以何种数量存在。因此，转铁蛋白被生命科学和医学领域的众多科学家所研究，其研究主要旨在揭示转铁蛋白在基因组中的表达水平、定位、结合位点、相互作用伙伴以及功能丧失的影响等信息。这些分析中的大多数严重依赖于抗体的使用，而抗体的生产成本高、困难，并且在伦理上存在问题。此外，涉及抗体的方法适用于固定的生物材料，这使得动态过程的重现具有挑战性。一种常见的解决方案是使用遗传技巧来表达一种与另一种蛋白质融合的TF，这种蛋白质反过来可以很容易地被检测到，因此被称为标签。然而，标签通常仅适用于单一或少数方法，或者标签较大，可能会干扰TF的功能。此外，标记的TF的额外表达可诱导伪影。此外，用于表达这种标记的构建体的工具仅可用于TF的子集。果蝇，黑腹果蝇，长期以来一直是研究基本生物过程的主要模式生物之一。它得益于一个庞大的、联系密切的世界范围的研究人员社区以及大量遗传和分子资源的可用性。重要的是，它允许在体内的调查，并创建新的工具的代转基因线是straightforward.Here，我们建议创建一个生物和bioinformatic资源，包括一个库的转基因果蝇TF基因已被标记在其原生基因组位点的飞线。我们还将建立一个数据库，其中包含以前未研究过的TF子集的表达和DNA结合的基本数据。我们的资源将减轻以前的标记蛋白质文库的许多缺点：1）我们的标记策略允许容易地交换标签，使得每个TF可用于各种下游应用; 2）我们将创建单个转基因系，其中TF的任一末端已被标记，这降低了标签干扰蛋白质功能的风险; 3）对于用替代末端表达的TF，我们将为每个末端创建一条线，以便研究每个TF的不同版本。我们将免费向研究界成员分发飞行路线，数据库也将免费使用。这些资源将为在任何可以想象的生物过程中选择研究任何果蝇TF的方法提供可能性，并使科学家能够轻松地在许多未研究的TF中识别潜在的候选者。我们希望我们的资源将大大推进基因调控领域的研究。