Drosophila models of human mitochondrial diseases

人类线粒体疾病的果蝇模型

基本信息

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
项目类别：
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

项目摘要

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条件有关，范围从代谢和退化范围过早衰老的疾病。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。一种方法了解线粒体疾病的生理病理学是创建动物模型，以详细说明潜在扰动的机械研究。果蝇是一种已建立的成本效益模型系统用于研究人类疾病，包括线粒体疾病。飞行模型的验证取决于两个关键集工具：扰动基因功能和识别蛋白质的试剂的试剂。而扰动试剂几乎所有14,000个果蝇基因都很容易获得，相对较少的试剂可检测蛋白质。我们的建议重点是生成试剂，这些试剂将作为一种资源来实现人类线粒体疾病的果蝇模型的表征和验证。我们将生成基于靶基因结构的两组试剂，用于检测由394高的蛋白质检测人线粒体疾病基因的置信果蝇直系同源物。对于第1组基因（AIM 1），其中所有同工型共享相同的C末端外显子，我们将在内源性的C末端插入纳米型表位基因使用基于CRISPR的基因组工程。我们选择了3'端以插入标签，将标签插入n 末端可能会干扰线粒体定位信号。然后可以是由此产生的蛋白质融合由现有的高亲和力纳米肌对纳米塔的认可。对于第2组基因（AIM 2），编码多种同工型，包括一些具有不同C端外显子的同工型，我们将使用蛋白质结构域常见对所有同工型筛选纳米生物体。这些试剂将与现有的RNAi转基因一起使用验证人线粒体疾病的苍蝇模型的线（AIM 3）。纳米体和飞股的资源随着纳米塔的“敲入”，内源基因将显着扩大可用试剂的范围蝇线粒体蛋白的检测和生化表征。