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是一种在脊椎动物进化过程中保守的运动神经元特异性microRNA， 人和小鼠的胎儿和成人下运动神经元。在人类和小鼠中有两种miR-218等位基因， 并且已经产生了miR-218-1和miR-218-2的无效等位基因。结果发现，缺乏这两种基因的胚胎 miR-218的等位基因正常发育，具有适当的运动神经元数量，然而，在最后一周， 在胚胎发育过程中，它们的运动神经元退化，其病理生理学类似于脊髓肌肉萎缩， 萎缩（SMA）和肌萎缩侧索硬化（ALS）。虽然ALS的运动退化发生在成年人身上， 而不是在miR-218突变小鼠中观察到的胎儿阶段，一些发现提供了进一步的间接支持， ALS和miR-218之间的联系：（1）基因表达研究，以确定在ALS中失调的microRNA。 散发性和家族性ALS反复检测到miR-218下调。(2)最近对ALS患者的分析 发现了一个miR-218突变的队列，表明miR-218水平或活性不足可能是一个风险因素 来治疗这种疾病。(3)许多与ALS相关的基因影响RNA代谢和微小RNA加工复合体， 包括TDP 43、FUS和C9 orf 72，提示ALS中miR-218下调的合理机制。（四） 在初步研究中，产生了miR-218的缺失突变，发现成年人中的缺失导致miR-218的缺失。 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 （1）确定在动物模型中遗传降低miR-218是否加速ALS病理学，和（2） 异位miR-218是否可以重新编程运动神经元中的正常遗传电路并减轻ALS 退化这项资助的目标是为ALS研究社区生产和提供miR-218试剂 加速对这种有前途的ALS治疗新候选药物的研究。目标1：浓度 将在小鼠系的等位基因系列中定义miR-218的水平。预计这些线路可用于 其他人则系统地与他们的ALS模型交叉，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）将来源于我们的miR-218等位基因系， 因为具有不同miR-218水平的运动神经元可以容易且有效地被 用于研究ALS的细胞/分子特征。的试剂 将通过将我们的小鼠品系存放在 杰克逊实验室储存库，并保持mES细胞系的专用储备用于分销。非 重叠的补充R21赠款也已提交，以表征受调控的遗传网络 通过miR-218，为了从机制上定义由其调节的运动神经元突触和存活模块， microRNA。