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pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA

contrast, no variation in the levels of let-7 miRNA or 5.8S and 5S rRNAs was observed. The exogenous expression of mature miRNA was further confirmed by solution hybridization using a probe specific for miR-124. Expression of this brain-specific miRNA was detected only in cells transfected with the CMV miR-124 vector (Figure 2B). Like Chen et al. [3], we found that native genomic sequences flanking each side of the predicted hairpin are required for correct processing of pri-miRNA into mature miRNA. Expression of shorter pri-miRNA transcripts lacking the flanking sequences resulted in accumulation of unprocessed pre-miRNA hairpins as determined by Northern blot analysis (data not shown).


High Level of Protein Expression

In addition to providing high-level expression of mature siRNA or miRNA, the CMV promoter is excellent for stable or transient expression of protein in a wide range of mammalian cells. To test the ability of the pSilencer 4.1-CMV vectors to drive high levels of mRNA and protein expression, vectors containing either a firefly luciferase cDNA or a chloramphenicol acetyltransferase (CAT) cDNA were prepared and transfected into HeLa cells. The close correlation between luciferase or CAT gene transcript levels and the enzymatic activity of their corresponding proteins have made these systems powerful genetic reporters for investigating transcriptional and post-transcriptional regulation in mammalian cells. As shown in Figure 3, high levels of reporter protein were readily detected in the transfected cells 24 or 48 hours after
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