miRNA Function-Reporter Expression Assay

Abstract

The detection of microRNAs (miRNAs) at single-cell resolution is crucial for acquiring knowledge about the role of these post-transcriptional regulators. Huttner and colleagues from the Max Planck Institute of Molecular Cell Biology and Genetics (Dresden, Germany) developed a miRNA Function-Reporter Expression assay for this purpose. It is a relatively simple and reliable system that allows the detection of miRNAs with cellular resolution in vivo without the need to generate transgenic animals (Biotechniques 41:727–732, 2006). The system is based on the acute administration of a dual-fluorescence GFP-reporter/mRFP-sensor (DFRS) plasmid for a specific miRNA into the organism of interest. In their DFRS plasmids, both GFP and mRFP are under the control of identical constitutive promoters. The GFP-reporter is used to identify the cells actually expressing the plasmid, given that the sensor-based strategy relies on the silencing of a transcript. The mRFP-sensor contained a 3′ untranslated region (3′UTR) with a tandem cassette complementary to the miRNA of interest. To establish a system allowing the monitoring of miRNA dynamics in defined cell lineages during mammalian embryonic development, the group explored the use of DFRS plasmids in conjunction with a combination of methods previously used to achieve acute expression of transgenes and RNA interference in developing mouse embryos. This combination consists of the topical release of nucleic acids in the proximity of a specific tissue of a mouse embryo developing either in culture or in utero and their delivery into this tissue by directed electroporation. This strategy provides a simple approach to study a specific miRNA in the tissue and cell lineage of interest.