Drosophila models of human mitochondrial diseases

人类线粒体疾病的果蝇模型

• 批准号: 10756280
• 负责人: NORBERT PERRIMON
• 金额: $ 86.73万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10756280
• 关键词: Affect; Affinity; Aging; Alpaca; Animal Model; Base Sequence; Biochemical; Biological Assay; Biological Models; Biomedical Research; Chimeric Proteins; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Complementary DNA; Defect; Degenerative Disorder; Detection; Diabetes Mellitus; Drosophila genus; Drug or chemical Tissue Distribution; Enhancement Technology; Epitopes; Exons; Functional disorder; Gene Structure; Genes; Genetic; Genome engineering; Heart failure; Human; Immunoprecipitation; Individual; Knock-in; Liver diseases; Mediating; Metabolic Diseases; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Modeling; Morphology; Muscle Weakness; Mutation; Nerve Degeneration; Organ; Orthologous Gene; Pathology; Patients; Phenotype; Premature aging syndrome; Protein Isoforms; Proteins; Protocols documentation; Public Domains; Publishing; R24; RNA Interference; Reagent; Reproducibility; Resource Development; Resources; Role; Seizures; Signal Transduction; Technology; Tertiary Protein Structure; Tissues; Transgenic Organisms; United States National Institutes of Health; Validation; cost effective; experimental study; fly; gene function; human disease; human model; in vivo; interest; kidney dysfunction; knock-down; nanobodies; response; tool

PROJECT SUMMARY / ABSTRACT Defects in mitochondria are associated with a spectrum of conditions, ranging from metabolic and degenerative diseases to premature aging. Mitochondrial defects are also associated with multiple organ defects, including muscular weakness, cardiac failure, diabetes, renal dysfunction, and hepatic disease. One approach to understanding the physiopathology of mitochondrial diseases is to create animal models that enable detailed mechanistic studies of the underlying perturbations. Drosophila is an established cost-effective model system for studying human diseases, including mitochondrial diseases. Validation of fly models relies on two critical sets of tools: reagents that perturb gene function and reagents that recognize proteins. While perturbation reagents are readily available for nearly all 14,000 Drosophila genes, there are relatively few reagents for detection of fly proteins. Our proposal focuses on generating reagents that will serve as a resource to enable the characterization and validation of Drosophila models of human mitochondrial diseases. We will generate two sets of reagents, based on the target gene structure, for the detection of proteins encoded by 394 high confidence Drosophila orthologs of human mitochondrial disease genes. For Group 1 genes (Aim 1), where all isoforms share the same C-terminus exon, we will insert a NanoTag epitope at the C-terminus of the endogenous gene using CRISPR-based genome engineering. We selected the 3’ end to insert the tag as inserting it at the N terminus could interfere with the mitochondrial localization signal. The resulting protein fusions can then be recognized by an existing high-affinity nanobody against the NanoTag. For Group 2 genes (Aim 2), which encode multiple isoforms, including some with different C-terminus exons, we will use a protein domain common to all isoforms to screen for nanobodies. These reagents will be used together with existing RNAi transgenic lines to validate fly models of human mitochondrial diseases (Aim 3). The resource of nanobodies and fly stocks with NanoTags ‘knocked-in’ to endogenous genes will dramatically expand the scope of available reagents for detection and biochemical characterization of fly mitochondrial proteins.

项目摘要/摘要 线粒体缺陷与一系列疾病有关，包括新陈代谢和退行性疾病。 疾病到过早衰老。线粒体缺陷也与多个器官缺陷有关，包括 肌肉无力、心力衰竭、糖尿病、肾功能障碍和肝病。一种方法是 了解线粒体疾病的生理病理学就是建立动物模型，使详细的 对潜在扰动的机械研究。果蝇是一种成熟的高性价比模型系统 用于研究人类疾病，包括线粒体疾病。苍蝇模型的验证依赖于两个关键集合 工具种类：干扰基因功能的试剂和识别蛋白质的试剂。而干扰试剂 几乎所有14,000个果蝇基因都可以轻易获得，但用于检测苍蝇的试剂相对较少 蛋白质。我们的建议侧重于生成试剂，这些试剂将作为一种资源，使 人类线粒体疾病果蝇模型的特征和验证。我们将生成 基于靶基因结构的两套试剂，用于检测394高编码蛋白 果蝇对人类线粒体疾病基因同源基因的信心。对于组1基因(目标1)，其中所有 异构体共享相同的C-末端外显子，我们将在内源性的C-末端插入一个NanoTag表位 基因使用基于CRISPR的基因组工程。我们选择3‘端来插入标签，就像在N处插入标签一样 末端可能干扰线粒体定位信号。由此产生的蛋白质融合可以 被现有的针对NanoTag的高亲和力纳米体所识别。对于组2基因(目标2)，它 编码多种异构体，包括一些带有不同C末端外显子的异构体，我们将使用一个共同的蛋白质结构域 所有的异构体来筛选纳米小体。这些试剂将与现有的RNAi转基因一起使用 用于验证人类线粒体疾病苍蝇模型的品系(目标3)。纳米体和苍蝇种群的资源 利用纳米标签“敲入”内源基因将极大地扩大可用试剂的范围 苍蝇线粒体蛋白的检测及生化特性研究。