A Plasmid-Based miRNA Sensor Library for Use in Mice

描述(申请人提供)：项目摘要/摘要：microRNAs(MiRNAs)是一种小的、核编码的单链RNA，参与调控基因的表达。直到最近才在哺乳动物中发现，利用战略克隆、生物信息学和系统发育分析，迄今已能够识别数百个哺乳动物miRNA基因。有证据表明，miRNAs在发育、分化、代谢和疾病等多种生物学过程中发挥作用。与其广泛的功能一致，计算机算法表明miRNAs可能调节基因组中多达三分之一的蛋白质编码基因的表达。然而，实验上只将靶基因分配给了少数miRNAs，而绝大多数miRNAs的确切功能尚不清楚。1解开单个miRNAs功能的方法是确定它们的表达模式。用于监测信使RNA表达的技术，如Northern blotting和微阵列分析，已经被用于监测miRNA的表达。然而，这些方法是劳动密集型和耗时的，并且miRNAs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，为了获得miRNAs，动物或细胞必须被摧毁，这使得时间进程研究变得更加困难和昂贵。在这项第一阶段的研究中，我们建议开发一种用于动物的系统，使研究人员能够随着时间的推移监测miRNAs的表达。该系统是基于这样一个事实，即在mRNA中放置精确匹配的miRNA结合位点会导致通过RNAi途径进行mRNA切割。与已知的所有小鼠miRNA完全匹配的miRNA结合位点将被放入3？一个非免疫原性的分泌型报告基因的非编码区。报告基因将被保存在一个含有在小鼠肝脏中高、长期表达所必需的元素的质粒DNA中。将使用流体动力尾静脉注射来完成质粒递送，这是一种有效地将质粒DNA递送到肝脏的简便方法。共传递表达不同的、非免疫原性的、分泌的、不受miRNA控制的报告基因的质粒将起到控制传递效率的变化的作用。使用这种方法，可以在一天内产生大量的miRNA传感器小鼠，任何品系的小鼠都可以用于实验。这些小鼠将允许研究人员简单地通过测量血清中存在的报告基因的数量来快速、轻松地监测任何感兴趣的miRNA的表达变化。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，而且还将用于监测miRNA表达随时间和不同治疗条件的变化。项目简介：microRNAs(MiRNAs)是调节基因表达的小RNA。哺乳动物物种中存在数百种不同类型的miRNAs。我们建议创建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。

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.