Tools for regulated expression control of miR-218

用于调控 miR-218 表达控制的工具

• 批准号: 10196829
• 负责人: SAMUEL L. PFAFF
• 金额: $ 19.24万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10196829
• 关键词: ALS pathology; ALS patients; Adult; Affect; Alleles; Amyotrophic Lateral Sclerosis; Animal Model; Applied Research; Attenuated; Basic Science; Benchmarking; Biological Assay; Biology; Birth; C9ORF72; Cell Line; Cells; Communities; Complex; DNA Databases; Data; Defect; Deposition; Disease; Distal; Down-Regulation; ES Cell Line; Embryo; Embryonic Development; Enterobacteria phage P1 Cre recombinase; Evolution; Functional disorder; Gene Expression; Generations; Genes; Genetic; Genotype; Glioblastoma; Glioma; Goals; Grant; Hand Strength; Health; Hindlimb; Human; In Vitro; Investigation; Karyotype; Laboratories; Life; Link; LoxP-flanked allele; Malignant neoplasm of central nervous system; MicroRNAs; Mining; Modeling; Molecular; Motor; Motor Neurons; Mouse Strains; Mus; Muscle; Mutant Strains Mice; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Pancreas; Pathology; Physiology; Play; Prognosis; Protocols documentation; PubMed; Publications; RNA metabolism; Reagent; Research; Research Personnel; Resources; Risk Factors; Role; Series; Signal Transduction; Spinal; Spinal Cord; Spinal Muscular Atrophy; Synapses; Tamoxifen; Testing; The Jackson Laboratory; Therapeutic; Therapeutic Agents; Transgenes; Transgenic Mice; Validation; Work; amyotrophic lateral sclerosis therapy; cell type; cohort; embryonic stem cell; familial amyotrophic lateral sclerosis; fetal; in vitro testing; in vivo; interest; medulloblastoma; neuromuscular; novel therapeutics; pathogen; pluripotency; programs; protein TDP-43; repository; spatiotemporal; therapeutic target; tool

MiR-218 is a motor neuron-specific microRNA conserved across vertebrate evolution that is expressed by both fetal and adult lower motor neurons in humans and mice. There are two alleles of miR-218 in humans and mice, and null alleles of both miR-218-1 and miR-218-2 have been generated. It was found that embryos lacking both alleles of miR-218 develop normally with proper motor neuron numbers, however during the final week of embryonic development their motor neurons degenerate with a pathophysiology that mimics spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Although motor degeneration in ALS occurs in adults rather than at fetal stages as observed in miR-218 mutant mice, several findings provide further indirect support for a connection between ALS and miR-218: (1) Gene expression studies to identify microRNAs dysregulated in sporadic and familial ALS repeatedly detect downregulation of miR-218. (2) A recent analysis of ALS patients found a cohort with mutations in miR-218, suggesting insufficient miR-218 levels or activity may be a risk factor for the disease. (3) Many ALS-linked genes affect RNA metabolism and microRNA processing complexes, including TDP43, FUS and C9orf72, suggesting a plausible mechanism for miR-218 downregulation in ALS. (4) In preliminary studies a floxed-mutation of miR-218 was generated and it was found that deletion in adults leads to neuromuscular pathology. These observations suggest several obvious research possibilities, including tests to: (1) determine whether genetically-lowering miR-218 accelerates ALS-pathology in animal models and (2) whether ectopic miR-218 can reprogram the normal genetic circuits in motor neurons and attenuate ALS degeneration. The goal of this grant is to generate and provide miR-218 reagents to the ALS research community to accelerate the investigation of this promising new candidate for ALS-therapy. In Aim 1, the concentration levels of miR-218 will be defined in an allelic series of mouse lines. It is anticipated that these lines can be used by others to cross to their ALS-models systematically to raise the lower miR-218 levels in vivo. In Aim 2 mouse embryonic stem cell lines (mESC) will be derived from our allelic series of miR-218 lines which is anticipated to have value in culture assays since motor neurons with different miR-218 levels can easily and efficiently be generated from mES lines for investigating the cellular/molecular features of ALS in vitro. The reagents generated in this grant will be made widely available to researchers by depositing our mouse strains in the Jackson Laboratories repository and by maintaining a dedicated stock of mES cell lines for distribution. non- overlapping complementary R21 grant has also been submitted to characterize the genetic networks regulated by miR-218, in order to define mechanistically the motor neuron synaptic and survival modules regulated by this microRNA.

MIR-218是一种运动神经元特异性microRNA，在脊椎动物进化过程中保守，由两者表达 胎儿和成人降低了人类和小鼠的运动神经元。在人类和小鼠中，miR-218有两个等位基因， MiR-218-1和miR-218-2均为空等位基因。研究发现，胚胎同时缺乏这两种功能 MiR-218的等位基因发育正常，运动神经元数量适当，但在 胚胎发育，他们的运动神经元退化，其病理生理学模仿脊髓肌肉 萎缩(SMA)和肌萎缩侧索硬化症(ALS)。尽管肌萎缩侧索硬化症的运动变性发生在成人 而不是像在miR-218突变小鼠中观察到的那样处于胎儿期，一些发现提供了进一步的间接支持 肌萎缩侧索硬化症和miR-218之间的联系：(1)基因表达研究，以确定 散发性和家族性ALS反复检测到miR-218的下调。(2)肌萎缩侧索硬化症患者的近期分析 发现一组miR-218基因突变，表明miR-218水平或活性不足可能是一个危险因素 治疗这种疾病。(3)许多ALS连锁基因影响RNA代谢和microRNA加工复合体， 包括TDP43、FUS和C9orf72，提示ALS中miR-218下调的可能机制。(4) 在初步研究中，产生了miR-218的花状突变，并发现在成年人中缺失导致 神经肌肉病理学。这些观察结果表明了几种明显的研究可能性，包括测试 目的：(1)确定基因降低的miR-218是否会加速动物模型的ALS病理；(2) 异位miR-218能否对运动神经元的正常遗传回路进行重新编程并减弱ALS 退化。这项资助的目标是为肌萎缩侧索硬化症研究社区生产和提供miR-218试剂。 以加快对这一有希望的ALS治疗新候选者的研究。在目标1中，浓度 MiR-218的水平将在一系列等位基因的小鼠中定义。预计这些线路可以使用 其他人系统地交叉到他们的ALS模型，以提高体内较低的miR-218水平。在Aim 2小鼠中 胚胎干细胞系(MESC)将从我们的miR-218等位基因系列衍生而来，预计将 在培养检测中有价值，因为不同miR-218水平的运动神经元可以容易和有效地 从MES系中产生，用于体外研究ALS的细胞/分子特征。试剂盒 通过将我们的小鼠品系存放在 杰克逊实验室的资源库，并维护MES细胞系的专用库存以供分配。非- 还提交了重叠的互补R21赠款，以表征受监管的遗传网络 通过miR-218，为了机械地定义受此调控的运动神经元突触和生存模块 微核糖核酸。