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是跨脊椎动物演化保守的运动神经元特异性microRNA，这两者都表达 人类和小鼠的胎儿和成人下运动神经元。人类和小鼠有两个miR-218等位基因， MiR-218-1和miR-218-2的无效等位基因均已产生。已经发现胚胎都缺乏 miR-218的等位基因正常发育有适当的运动神经元数，但是在 胚胎发育的运动神经元通过模拟脊柱肌肉的病理生理学退化 萎缩（SMA）和肌萎缩性侧索硬化症（ALS）。尽管ALS的运动变性发生在成年人中 几个发现提供了进一步的间接支持，而不是在miR-218突变小鼠中观察到的胎儿阶段 对于ALS与miR-218之间的联系：（1）基因表达研究，以鉴定在 零星和家族性ALS反复检测到miR-218的下调。 （2）最近对ALS患者的分析 在miR-218中发现了一个与突变的队列，这表明miR-218水平不足或活动可能是危险因素 为了疾病。 （3）许多ALS连接基因影响RNA代谢和microRNA加工复合物， 包括TDP43，FUS和C9ORF72，这表明了ALS中miR-218下调的合理机制。 （4） 在初步研究中 致神经肌肉病理学。这些观察结果表明了一些明显的研究可能性，包括测试 至：（1）确定遗传降低的miR-218是否在动物模型中加速了ALS-PATHOLOGY和（2） 异位miR-218是否可以在运动神经元中重新编程正常遗传回路并减弱ALS 退化。这笔赠款的目的是生成并为ALS研究社区提供miR-218试剂 加快对这一有希望的新候选ALS治疗的调查。在AIM 1中，集中度 miR-218的水平将在等位基因系列小鼠系中定义。预计可以使用这些行 其他人则系统地跨越其ALS模型，以提高体内miR-218水平。在AIM 2鼠标中 胚胎干细胞系（MESC）将源自我们的等位基因MiR-218系列，预计将会 在培养测定中具有价值，因为具有不同miR-218水平的运动神经元很容易有效地是 由MES线产生，用于研究ALS体外的细胞/分子特征。试剂 通过将我们的鼠标菌株存入到 Jackson Laboratories存储库，并维护MES细胞系的专用库存以进行分发。非 重叠的互补R21赠款也已提交以表征受监管的遗传网络 由miR-218，为了机械定义运动神经元突触和生存模块。 microRNA。