Efficient Gene Silencing
To demonstrate the utility of the pSilencer CMV vectors in RNA interference experiments, a pSilencer 4.1-CMV vector was engineered to express a hairpin siRNA targeting GAPDH. This construct was then transfected into HeLa cells. As shown in Figure 1, the level of GAPDH protein was reduced by ~80% two days after transfection. This suggests that the hairpin siRNA was efficiently expressed and correctly processed in the cell to generate a mature, functional siRNA. Long-term antibiotic selection of the transfected cells produced cell lines exhibiting reduced levels of GAPDH mRNA and protein for over 4 months (see 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 GAPDH mRNA (CMV-Gap) or a scrambled sequence (CMV-Scr). (A) Immunostaining of HeLa cells two days after transfection showing reduction of GAPDH levels in cells transfected with CMV-Gap. Green: GAPDH protein. Blue: DAPI stained nuclei. (B) Relative levels of GAPDH expression compared with HeLa cells not transfected (NT).
Figure 2. Expression of miRNA Using pSilencer 4.1-CMV hygro. pSilencer 4.1-CMV was engineered to express mir-16 or mir-124 pri-miRNA. (A) Schematic representation of the mir-16 and mir-124 transcripts. Each transcript included the predicted hairpin at its center and flanking genomic sequences of various sizes. (B) Two days after transfection into HeLa cells, total RNA was isolated with the mirVana miRNA Isolation Kit and 1 g of total RNA was analyzed on a denaturing agarose gel. A Northern blot was used to detect let-7 miRNA, 5.8S and 5S rRNA while miR-16 and miR-124 miRNA were detected by solution hybridization with the mirVana miRNA Detection Kit. All probes were labeled and purified with the mirVana Probe & Marker Kit. NT = not transfected.
Two days after transfection, total RNA was isolated from the cell cultures using the mirVana miRNA Isolation Kit, which provides quantitative yield of small RNAs, including miRNA, siRNA, tRNA, and 5S/5.8S rRNAs. Analysis of miRNA expression by solution hybridization assay with the mirVana miRNA Detection Kit showed a significant overexpression of miR-16 in cells transfected with the pSilencer 4.1-CMV miR-16 vector (Figure 2B). In 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. , 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 transfection. The presence of a hygromycin, neomycin, or puromycin antibiotic resistance genes on each pSilencer 4.1-CMV vector further allows for selection of stable cell lines constitutively expressing these reporters.
Figure 3. Expression of mRNA Using pSilencer 4.1-CMV puro. HeLa cells were transfected in triplicate with pSilencer 4.1-CMV vectors carrying either a firefly luciferase cDNA or a CAT cDNA. (A) Relative luciferase activity 24 hours after transfection. (B) Relative CAT activity 48 hours after transfection. NT = not transfected. RU = relative units.
The pSilencer 4.1-CMV vectors are provided linearized and ready for ligation. They are part of Ambion's extensive line of plasmid and viral expression systems. For more information or to determine which vector best suits your needs, please visit www.ambion.com/prod/psilencer.
Cat# Product Name Size 1552 mirVana miRNA Detection Kit 100 rxns 1554 mirVana Probe & Marker Kit 30 rxns + 10 marker rxns 1560 mirVana miRNA Isolation Kit up to 40 purifications 5775 pSilencer 4.1-CMV puro 20 rxns 5777 pSilencer 4.1-CMV hygro 20 rxns 5779 pSilencer 4.1-CMV neo 20 rxns