pSilencer™
siRNA Exression Vector

In early 2002, Ambion began providing the Silencer™ siRNA Construction Kit and Silencer™ Custom siRNA Oligonucleotide Synthesis Service for generating siRNAs for mammalian gene silencing experiments. Now Ambion introduces the pSilencer™ 1.0-U6 Vector for expression of your choice of siRNAs in mammalian cells.

Expression of siRNA in Vivo Elicits Gene Silencing
RNA interference (RNAi) is now being used routinely in mammalian cells to study the functional consequences of reducing the expression of specific genes. RNAi is induced by transfecting small interfering RNAs (siRNAs), comprising double-stranded RNA molecules ~21 nt in length with 2 nt 3' overhangs (1), or hairpin-forming 45-50mer RNA molecules (2), that are complementary to the gene of interest. Several groups have recently shown that RNAi can also been induced by transfecting plasmids that express siRNAs within cells (2-7). Although the details vary, most of the groups developed plasmids with a sequence encoding a small hairpin RNA (shRNA) under the control of an RNA Polymerase III (Pol III) promoter. The U6 and H1 Pol III promoters were chosen because Pol III initiates synthesis at a defined distance from these promoters and terminates when a string of 4-5 uridines is encountered. When transfected into mammalian cells, siRNA expression plasmids and have been shown to reduce the levels of both exogenous and endogenous gene products (2-7). Although they require more effort to prepare than chemically synthesized or in vitro transcribed siRNAs, the siRNA vectors can provide longer term reduction in target gene expression when coexpressed with a selectable marker (3).

An Expression Vector to Silence Your Genes of Interest
Ambion now offers the pSilencer 1.0-U6 Vector to facilitate plasmid-based siRNA experiments. pSilencer 1.0-U6 contains a U6 Pol III promoter and sequence elements for cloning and bacterial replication. This vector was developed by Sui and colleagues at Harvard Medical School and has been successfully used to knock down expression of cdk-2 and lamin A/C in HeLa, H1299, U-2 OS and C-33A (cdk-2 only) cells (4).

Figure 1 shows the use of pSilencer 1.0-U6 to reduce the expression of GAPDH in HeLa cells. The pSilencer 1.0-U6 vector containing a 55-mer DNA insert encoding an RNA hairpin directed to GAPDH reduced GAPDH protein expression by 85%. Transfecting siRNA targeting the same sequence also resulted in significant reduction in GAPDH protein levels, suggesting that similar target sequences can be used with exogenously and endogenously produced siRNA (8).

Figure 1. Plasmid Vector Expression of siRNA Cause Gene Silencing. HeLa cells were plated at 60,000 cells per well in a 24 well tissue culture plate containing glass cover slips and transfected 24 hours later using 0.8 mg of pSilencer™ 1.0-U6 GAPDH plasmid. The cells were subsequently split and replated onto glass cover slips to obtain appropriate density. A. Immunofluorescence for GAPDH protein, 96 hours post transfection. B. Fluorescent signal, standard deviation, and cell number of the transfected and non-transfected samples, quantitated using MetaMorph software (Nikon). The fluorescent signal was normalized to cell number and the percent of non-transfected levels was calculated using Microsoft Excel. C. The graph shows an 85% reduction of GAPDH protein levels in cells transfected with the pSilencer 1.0-U6 GAPDH plasmid when compared to non-transfected levels.

To use the pSilencer 1.0-U6 Vector, linearize the vector with Apa I and EcoR I and design an appropriate ~55 bp insert sequence. The double-stranded DNA insert should include 4 nucleotide overhangs complementary to the Apa I and EcoR I restriction sites, as well as the sense and antisense sequences of the desired siRNA separated by a small loop sequence (Figure 2). This double-stranded DNA insert is then ligated into the linearized vector and introduced into E. coli cells. The resulting plasmid is produced in E. coli, purified and then transfected into mammalian cells.

The pSilencer 1.0-U6 Vector is available as a circular plasmid and is supplied at a concentration of 0.5 mg/ml.

Figure 2. Schematic of pSilencer™ 1.0 U6. A. Plasmid map. B. Example insert for expressing an siRNA. The loop sequence and length can be varied as desired (2-7).

The pSilencer 1.0-U6 Vector is the subject of a US Patent Application and is sold under licensing arrangement with Harvard Medical School.

REFERENCES

1. Elbashir, SM, Harborth, J, Lendeckel, W, Yalcin, A, Weber, K, and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.

2. Paddison, PJ, Caudy, AA, Bernstein, E, Hannon, GJ and Conklin DS. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Development 16:948-958.

3. Brummelkamp, TR, Bernards, R, and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550-553.

4. Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC, and Shi Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 99(8): 5515-5520.

5. Miyagishi M & Taira K (2002) "U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells," Nature Biotechnology 19: 497-500.

6. Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, Rossi J (2001) "Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells," Nature Biotechnology 19: 500-505.

7. Paul CP, Good PD, Winer I, Engelke DR (2002) "Effective Expression of Small Interfering RNA in human cells," Nature Biotechnology 20: 505-508.

8. Brown D, Jarvis R, Pallotta V, Byrom M, Ford L (2002) RNA Interference in Mammalian Cell Culture: Design, Execution and Analysis of the siRNA effect. Ambion TechNotes 9(1): 3-5.

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