A Plasmid-Based miRNA Sensor Library for Use in Mice

用于小鼠的基于质粒的 miRNA 传感器库

• 批准号: 7157106
• 负责人: David Lawrence Lewis
• 金额: $ 19.76万
• 依托单位: MIRUS BIO CORPORATION
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-07 至 2008-08-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7157106
• 关键词: RNA interference; alkaline phosphatase; antisense nucleic acid; bioengineering /biomedical engineering; blood tests; gene expression; gene targeting; genetic library; genetic regulation; injection /infusion; laboratory mouse; liver; longitudinal animal study; messenger RNA; method development; microRNAs; molecular genetics; plasmids; reporter genes; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Project Summary/Abstract: MicroRNAs (miRNAs) are small, nuclear encoded, single-stranded RNAs involved in regulating gene expression. Only recently discovered in mammals, use of strategic cloning, and bioinformatics and phylogenetic analyses have enabled the identification of a few hundred mammalian miRNA genes to date. There is evidence that miRNAs play roles in a wide variety of biological processes including development, differentiation, metabolism and disease. Consistent with their wide-ranging function, computer algorithms indicate that miRNAs may regulate expression of up to 1/3rd of protein-coding genes in the genome. However, target genes have been experimentally assigned to only a handful of miRNAs and the precise functions of the vast majority of miRNAs remain unknown. 1 approach used to unravel the function of individual miRNAs is to determine their expression patterns. Techniques for monitoring messenger RNA expression such as Northern blotting and microarray analysis have been adapted to monitor miRNA expression. However, these approaches are labor intensive and time-consuming, and the small size of miRNAs makes these hybridization-based methods of detection technically challenging. In addition, the animals or cells must be destroyed in order to harvest the miRNAs, making time course studies more difficult and expensive. In this Phase I study, we propose to develop a system to be used in animals that will allow an investigator to monitor the expression of miRNAs over time. The system is based on the fact that placement of an exact-match miRNA binding site in an mRNA results in mRNA cleavage via the RNAi pathway. Exact match miRNA binding sites for all known mouse miRNAs will be placed into the 3? UTR of a non-immunogenic, secreted reporter gene. The reporter gene will be harbored in a plasmid DNA that contains elements necessary for high, long-term expression in mouse liver. Plasmid delivery will be accomplished using hydrodynamic tail vein injection, a facile method for efficient delivery of plasmid DNA to liver. Co-delivery of a plasmid expressing a different, non-immunogenic, secreted reporter gene not under miRNA control will function as a control for variations in delivery efficiency. Using this method, large numbers of miRNA sensor mice, of any strain, can be generated in a single day for use in an experiment. These mice will allow the investigator to quickly and easily monitor changes in expression of any miRNA of interest simply by measuring the amount of reporter gene present in the serum. This system will not only allow the investigator to determine if a particular miRNA is functional in the liver, but will also be useful for monitoring changes in miRNA expression over time and under different treatment conditions. Project Narrative: MicroRNAs (miRNAs) are small RNAs that regulate gene expression. There are hundreds of different types of miRNAs present in mammalian species. We propose to create a blood-based reporter system that will allow an investigator to determine if a miRNA of interest is expressed in the liver.

描述（由申请人提供）：项目摘要/摘要：微小RNA（miRNAs）是小的、核编码的、参与调节基因表达的单链RNA。直到最近才在哺乳动物中发现，使用战略克隆，生物信息学和系统发育分析，使数百个哺乳动物的miRNA基因的鉴定。有证据表明，miRNAs在包括发育、分化、代谢和疾病在内的多种生物过程中发挥作用。与它们广泛的功能一致，计算机算法表明，miRNA可以调节基因组中多达1/3的蛋白质编码基因的表达。然而，靶基因在实验上仅被分配给少数miRNAs，并且绝大多数miRNAs的精确功能仍然未知。用于揭示单个miRNAs功能的一种方法是确定它们的表达模式。用于监测信使RNA表达的技术，例如北方印迹和微阵列分析，已被用于监测miRNA表达。然而，这些方法是劳动密集型和耗时的，并且miRNA的小尺寸使得这些基于杂交的检测方法在技术上具有挑战性。此外，动物或细胞必须被破坏才能收获miRNA，这使得时间过程研究更加困难和昂贵。在这项I期研究中，我们建议开发一种用于动物的系统，使研究人员能够监测miRNA随时间的表达。该系统是基于这样的事实，即在mRNA中放置精确匹配的miRNA结合位点导致mRNA通过RNAi途径切割。将所有已知小鼠miRNA的精确匹配miRNA结合位点置于3？非免疫原性分泌报告基因的UTR。报告基因将隐藏在质粒DNA中，该质粒DNA含有在小鼠肝脏中长期高表达所需的元件。质粒递送将使用流体动力学尾静脉注射完成，这是一种将质粒DNA有效递送至肝脏的简便方法。表达不同的、非免疫原性的、分泌的报告基因的质粒的共递送不受miRNA控制，将用作递送效率变化的对照。使用这种方法，可以在一天内产生大量的任何品系的miRNA传感器小鼠用于实验。这些小鼠将允许研究者简单地通过测量血清中存在的报告基因的量来快速且容易地监测任何感兴趣的miRNA表达的变化。该系统不仅允许研究人员确定特定的miRNA是否在肝脏中起作用，而且还可用于监测miRNA表达随时间和不同治疗条件的变化。MicroRNA（miRNAs）是一种调节基因表达的小分子RNA。在哺乳动物物种中存在数百种不同类型的miRNA。我们建议建立一个基于血液的报告系统，使研究人员能够确定感兴趣的miRNA是否在肝脏中表达。