A Plasmid-Based miRNA Sensor Library for Use in Mice

点击翻译按钮获取中文摘要

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.