pmR-mCherry is a mammalian expression vector designed to constitutively express a microRNA of interest. Transfected cells can be identified by the coexpression of mCherry, a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). Coexpression of mCherry and your microRNA of interest allows easy monitoring and/or selection of microRNA-expressing cells by fluorescence microscopy or flow cytometry. The excitation and emission maxima of the native mCherry protein are 587 nm and 610 nm, respectively.

The pmR-mCherry multiple cloning site (MCS) is positioned in the 3’UTR, downstream of the mCherry coding sequence. Expression of mCherry and microRNA precursors cloned into the MCS is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE), located just upstream of the mCherry sequence. Both the fluorescent protein and the microRNA are expressed from a single mRNA transcript, which is cleaved by Drosha and Dicer to generate the mature microRNA.

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A small genomic fragment containing the precursor of the microRNA of interest must be isolated and cloned into pmR-mCherry. This is most easily accomplished by PCR amplification from genomic DNA. We recommend including 100-300 bp of genomic DNA flanking the actual microRNA precursor to ensure efficient processing by Drosha. The orientation of the cloned microRNA precursor should be the same as that of the mCherry transcript. The sequence of the microRNA precursor and flanking genomic DNA can be obtained from a number of public databases including GenBank (http://www.ncbi.nlm.nih.gov/) and EMBL-Bank (http://www. ebi.ac.uk/embl/). The UCSC Genome Bioinformatics Site (http://genome.ucsc.edu/) hosts an easy-to-navigate genomic database with tracks for microRNAs. The Sanger Institute hosts miRBase, a compilation of known microRNA sequences (http://microrna.sanger.ac.uk/).

The pmR-mCherry vector can be transfected into mammalian cells using any standard transfection method. If desired, stable transfectants can be selected using G418. Overexpressed microRNA can be detected using Clontech’s Mir-X™ miRNA qRT-PCR SYBR® Kit (Cat. Nos. 638314 and 638316). For Western analysis, the mCherry protein can be detected using either the Living Colors® DsRed Polyclonal Antibody (Cat. No. 632496) or the Monoclonal Antibody (Cat. Nos. 632392 and 632393).