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.

项目总结/摘要 线粒体缺陷与一系列疾病有关，从代谢性和退行性疾病， 疾病，过早衰老。线粒体缺陷也与多器官缺陷有关，包括 肌无力、心力衰竭、糖尿病、肾功能不全和肝病。的一种方法 了解线粒体疾病的生理病理学是创造动物模型， 对潜在扰动的机理研究。果蝇是一种已建立的具有成本效益的模型系统 用于研究人类疾病，包括线粒体疾病。飞行模型的验证依赖于两个关键集 工具：干扰基因功能的试剂和识别蛋白质的试剂。当微扰试剂 几乎所有14，000种果蝇基因都很容易获得，但用于检测果蝇的试剂相对较少。 proteins.我们的建议集中在产生试剂，这些试剂将作为资源，以使得能够进行化学反应。 人类线粒体疾病的果蝇模型的表征和验证。我们将产生 两套试剂，基于靶基因结构，用于检测由394高 人类线粒体疾病基因的果蝇直系同源物。对于组1基因（Aim 1），其中所有 如果内源性同种型共享相同的C-末端外显子，我们将在内源性同种型的C-末端插入NanoTag表位。 使用基于CRISPR的基因组工程。我们选择3'端插入标签，因为在N端插入标签 末端可能干扰线粒体定位信号。然后可以将所得的蛋白质融合物 被现有的针对纳米标签的高亲和力纳米抗体识别。对于第2组基因（Aim 2）， 编码多种亚型，包括一些具有不同C-末端外显子，我们将使用一个蛋白质结构域共同 来筛选纳米抗体。这些试剂将与现有的RNAi转基因生物一起使用。 线，以验证人类线粒体疾病的苍蝇模型（目标3）。纳米抗体和蝇类资源 随着NanoTags '敲入'内源基因将大大扩大可用试剂的范围， 果蝇线粒体蛋白的检测和生化特性。