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

om/techlib/tn/112/16.html">Drive Long-Term Silencing with CMV Vectors).


Expression of Mature miRNA

Expression of hairpin siRNAs for gene silencing is only one application of the pSilencer 4.1-CMV vectors; these vectors can also be used to express miRNA. miRNAs are endogenous, single-stranded small RNAs that are believed to play critical roles in eukaryotic development by controlling post-transcriptional gene expression. Like siRNAs, miRNAs are expressed as longer hairpin molecules. These primary transcripts (pri-miRNAs) are then cleaved in the nucleus by the Drosha protein to release 60-70 nt pre-miRNA hairpins. Pre-miRNAs are exported to the cytoplasm by an Exportin 5-dependent mechanism where they are processed by Dicer to generate mature 21-24 nt miRNAs [1, 2].

To evaluate whether pSilencer 4.1-CMV vectors could express pri-miRNA hairpins that could be processed by the cellular machinery to mature miRNA, genomic DNA fragments containing either the predicted mir-16 or mir-124 pri-miRNA hairpins were cloned in pSilencer 4.1-CMV hygro vectors. These plasmid constructs were then transfected into HeLa cells where they were expected to drive expression of the 180 nt mir-16 pri-miRNA transcript or the 150 nt mir-124 pri-miRNA (Figure 2A).

Figure 1. siRNA-mediated Knockdown of GAPDH Using pSilencer 4.1-CMV puro. HeLa cells were not transfected (NT) or transfected with pSilencer 4.1-CMV puro vectors engineered to express an siRNA targeting GA
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