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GENE SILENCING WITH PCR PRODUCTS

siRNA Expression Cassettes


siRNA expression vectors offer enormous opportunities to study gene silencing. Unfortunately, the preparation of siRNA vectors is labor and time intensive. Ambion's new Silencer Express siRNA Expression Cassette Kits provide a rapid PCR-based method for preparing siRNA expression cassettes (SECs) that can be introduced into cells to induce gene silencing without prior cloning or sequencing. Because they are easy to prepare, SECs are ideal for screening target sites. Once effective SECs are identified, they can be subcloned into an appropriate vector for
subsequent gene silencing studies.


siRNA Expression Cassettes: How They Work
The use of PCR products to express siRNAs in mammalian cells was recently described by Castanotto and colleagues (1). These PCR products, referred to as siRNA Expression Cassettes (SECs), contain an RNA Polymerase III (Pol III) promoter adjacent to a hairpin siRNA template and an RNA Pol III transcription termination site. When transfected into mammalian cells, the SECs are transcribed by endogenous RNA Pol III to generate a small hairpin siRNA with several U's at the 3' end. The hairpin is thought to be processed by the dsRNA-specific Dicer enzyme (2) to generate functional siRNAs, which can enter the RNAi pathway and reduce the expression of a target gene. Figures 14 show examples of gene silencing experiments performed using the SEC approach.

Figure 1. siRNA Expression Cassette-Mediated Reduction
in c-fos Expression.
siRNA Expression Cassettes encoding a negative control or c-fos-specific hairpin siRNA were transfected into HeLa cells. 72 hours post-transfection, the cells were assessed using nuclear staining with DAPI and immunofluorescence using a c-FOS antibody detected with a fluorescein labeled secondary antibody.

Figure 2. siRNA Expression Cassette-Mediated Reduction in GAPDH Expression. siRNA Expression Cassettes encoding a negative control or GAPDH-specific hairpin siRNA were transfected into HeLa cells. 72 hours post-transfection, the transfected cells and a non-transfected control sample (NT) were assessed using nuclear staining with DAPI and immunofluorescence using a GAPDH-specific antibody. The DAPI and immunofluorescence images are overlaid in the figures.

Figure 3. Variable Reduction in Target Gene Expression Using SECs with Different Promoters. siRNA Expression Cassettes featuring the mouse U6 (Mo-U6), human U6 (Hu-U6), and human H1 (Hu-H1) promoters and encoding a c-fos-specific hairpin siRNA were transfected into HeLa cells. 72 hours post-transfection, the cells were assessed using nuclear staining with DAPI and immunofluorescence using a c-FOS antibody. Non-transfected cells (NT) as well as cells transfected with an SEC expressing a negative control siRNA (scramble) demonstrate wild-type levels of c-FOS. The relative level of reduction in gene expression was quantified and is provided in the bar graph.

Figure 4. Reduction in -actin Expression Using SECs with Different Promoters. siRNA Expression Cassettes featuring the mous e U6 (Mo-U6), human U6 (Hu-U6), and human H1 (Hu-H1) promoters and encoding a -actin-specific hairpin siRNA were transfected into HeLa cells. 72 hours post-transfection, the cells were assessed using nuclear staining with DAPI and immunofluorescence using a -actin antibody. Non-transfected cells (NT) as well as cells transfected with an SEC expressing a negative control siRNA (scramble) provide wild-type levels of -actin. The relative level of reduction in gene expression was quantified and is provided in the bar graph.


Advantages of siRNA Expression Cassettes
In contrast to siRNA expression vectors, which require cloning and sequencing prior to use and can take 1-2 weeks to prepare, SECs can be prepared in less than a day. SECs thus provide an excellent screening tool to find the most effective siRNA sequence, or to identify the best combination of promoter and siRNA sequence in the experimental system being used. In fact, SECs provide the perfect complement to siRNA expression vectors. By incorporating restriction sites at their ends, SECs found to effectively elicit gene silencing can be readily cloned into a plasmid to create an siRNA expression vector. The siRNA expression vector can then be used for stable expression and long term studies.


Silencer Express Kits
Ambion's Silencer Express Kits provide the reagents necessary to prepare SECs with the Human H1, Human U6, or Mouse U6 promoters. Each kit includes a DNA fragment containing one of the three promoters, PCR primers specific to the 5' and 3' termini of the SECs, purification reagents to prepare SECs for transfection, and siRNA template oligonucleotides that can be used to generate positive and negative control SECs for siRNA studies. Both primers have modified ends to enhance SEC stability in mammalian cells (Figure 5). To facilitate cloning of the PCR products after testing, the 5' primer encodes an EcoR I restriction site and the 3' primer contains a Hind III restriction site. Ready-to-use pSEC receptor vectors with selectable markers will be available from Ambion soon. See the RNA Interference Resource at www.ambion.com/RNAi for more details.

Figure 5. Modifications in the Primers Provided with the Silencer Express Kit Produce siRNA Expression Cassettes (SECs) with Greater Stability in Cells. SECs designed to target GAPDH were prepared using the Silencer Express procedure from primers either with or without modifications to enhance stability. The SECs were transfected into 293 cells and RNA from the cells was analyzed after the indicated number of days. RNA was isolated with the RNAqueous Kit and then GAPDH mRNA levels were assessed by Northern blot using the NorthernMax-Gly Kit. The data are expressed as a percentage of GAPDH mRNA found in cells transfected with an SEC expressing a negative control (NC) siRNA. The modified SECs demonstrated a greater level of gene knockdown after 6 and 9 days. PCR primers contained in the Silencer Express Kits include the modifications used in this experiment.


Preparing SECs with the Silencer Expres s Kits is a simple, three-step process: (I) one or two oligonucleotides encoding the siRNA sequence are designed and ordered; (II) the oligonucleotide(s) are used as primers in one or more PCRs with the included RNA Pol III promoter-containing DNA template; and (III) the resulting PCR product is column-purified (see Overview of Silencer Express Procedure for details of the procedure). The SECs are then transfected into cells using standard DNA transfection agents. Transfection conditions are similar, but not identical, to vectors and will require some optimization.

The 3' ends of the three different promoter elements provided in the Silencer Express Kits share a common twenty-nucleotide sequence (see RNA Pol III Promoter Structure for details). This domain serves as the priming site for appending siRNA templates during PCR. Because the sequence is common, a single siRNA template oligonucleotide can be used with any of the three Silencer Express Kits. The benefit is that different promoters can be tested for their capacity to express active siRNAs in the cells being used. Based on published reports, the activities of the different promoters are likely to vary from cell type to cell type (3). The localization of expressed RNA is also likely to vary with cell type and with the RNA pol III promoter used (3). To optimize siRNA expression, we find it beneficial to create SECs with at l east two different promoters and transfect them into the cells being targeted for gene knockdown (Figures 3, 4, 6, and 7). The promoter that is more effective for the siRNA and cell type will provide greater levels of gene silencing.


SEC Validation
The Silencer Express Kits have been used to create SECs targeting to GAPDH, c-fos, cyclophilin, PKC-alpha, -actin, and Green Fluorescent Protein (GFP). In all cases, SECs were able to reduce target gene expression. Figures 14 and 6 show several examples. Furthermore, SECs targeting GAPDH were functional in HeLa, 293, Cos7, Du145, HepG2, NIH3T3, MCF7 and A549 cells, confirming that PCR products can be transfected and transcribed in different cell types. Because they are simple to prepare, SECs provide an ideal way to screen potential siRNA sequences for the best siRNA sequence-promoter combination for DNA-based RNAi experiments in diverse cell lines. For additional information and data, click here.

Figure 6. Effectiveness of RNA Pol III Promoters with Altered 3' Ends. siRNA Expression Cassettes featuring the human U6 (Hu-U6) and human H1 (Hu-H1) promoters and expressing Negative Control (scrambled), GAPDH-, and cyclophilin-specific hairpin siRNAs were transfected into 293 cells. 72 hours post-transfection, the cells were processed to recover total RNA. Approximately 500 ng of total RNA from each sample was assessed by Northern analysis. Radiolabeled RNA probes targeting 28S rRNA, GAPDH, and cyclophilin were used to detect target genes and normalize levels of input RNA. Films were exposed to t he blots for 16 hours with a single intensifying screen.

Figure 7. Schematic of Preparing SECs using Two siRNA Template Oligonucleotides.


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Ordering Information
Cat# Product Name Size 1680 Silencer Express (Human H1) 20 rxns 1681 Silencer Express (Human U6) 20 rxns 1682 Silencer Express (Mouse U6) 20 rxns 2050 SuperTaq Polymerase (Cloned) 5 U/l 50 U 2052 SuperTaq Polymerase (Cloned) 5 U/l 250 U 5772 pSEC hygro 20 rxns 5773 pSEC puro 20 rxns 5774 pSEC neo 20 rxns
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