More siRNA Vectors for RNA Interference

Small Interfering RNAs (siRNAs) are short, double-stranded RNA molecules that can target complementary mRNAs for degradation via a cellular process termed RNA interference (RNAi) (Elbashir et al., 2001a). Recent publications have described the use of vectors expressing functional siRNAs to enable long-term knockdown studies (for a review, see Tuschl, 2002). Two different RNA Polymerase III promoters, the well-characterized U6 and H1 promoters, have been used in these vectors to drive the expression of siRNAs (Sui et al., 2002, Brummelkamp et al., 2002). Below we discuss the reasons why these two promoters were chosen and introduce two new siRNA expression vectors for RNAi experiments in mammalian cells.

The selection of RNA Polymerase III to drive siRNA expression derives from the facts that it expresses relatively large amounts of small RNAs in mammalian cells and that it terminates transcription upon incorporating a string of 3-6 uridines. The latter feature is especially important due to the apparent requirement that siRNAs have defined 3' termini that hybridize to an mRNA target (Elbashir et al. 2001b). Type III promoters have been selected for siRNA vector design because they are relatively simple and they lie completely upstream of the sequence being transcribed. This eliminates any need to include promoter sequence in the siRNA.

The sequences of the U6 and H1 promoters are very distinct, with the U6 promoter stretching across several hundred base pairs (Kunkel 1989) and the H1 promoter barely exceeding 100 (Myslinski et al. 2001). The amount and location of siRNAs expressed from these two promoters is expected to vary from cell type to cell type. In studies comparing the expression of small RNAs under the control of the U6 promoter and several other RNA Pol III promoters, it was found that RNA synthesis can vary by orders of magnitude between the promoters depending on the cell line being used (Ilves et al. 1996). Furthermore, localization studies have shown that different RNA Pol III promoters generate small RNAs that localize to different sub-regions of cells (Ilves et al. 1996). The expression and localization of siRNAs will undoubtedly affect the success of RNAi studies in mammalian cells, making it important to test siRNAs under the control of both promoters.

In July 2002, Ambion introduced pSilencer 1.0-U6, which contains the mouse U6 promoter (Figure 1A).

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Figure 1. pSilencer™ siRNA Expression Vectors; Maps and siRNA Design. For each target gene two complementary 55-60 mer oligonucleotides must be prepared. The oligonucleotides should encode the 19 mer hairpin sequences specific to the mRNA target, a loop sequence separating the two complementary domains, and a polythymidine tract to terminate transcription by RNA Pol III. The insert design shown above is specific for the pSilencer 2.0-U6 and 3.0-H1 Expression Vectors and contains the overhanging 5' ends to facilitate efficient and directional cloning into these plasmids. The insert for pSilencer 1.0-U6 would contain the appropriate end sequences for cloning into the Apa I and EcoR I sites. Early indications suggest that a great deal of latitude is available in the design of the loop; here we provide our default loop sequence that we find works well.

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 (Sui et al., 2002). Ambion's latest pSilencer siRNA expression vectors, pSilencer 2.0-U6 and pSilencer 3.0-H1, feature two different RNA Polymerase III promoters (Figures 1B and 1C). pSilencer 2.0-U6 contains the human U6 promoter that has been used extensively to express siRNAs and ribozymes in mammalian cells. pSilencer 3.0-H1 features the H1 RNA promoter (a component of RNase P). The pSilencer 2.0-U6 and 3.0-H1 siRNA expression vectors are linearized with two different restriction enzymes to facilitate directional cloning. The linearized vectors are purified to eliminate religation of the vector with the digested fragment. Complementary oligonucleotides encoding siRNA sequences can be readily ligated into the vectors and used to transform E. coli to generate plasmids for siRNA studies (see "Selecting siRNA Sequences to Incorporate into the pSilencer Vectors"). The overhangs are the same for both the pSilencer 2.0-U6 and 3.0-H1 vectors (but are distinct from the linearized pSilencer 1.0-U6 vector); making it possible to subclone the same siRNA insert into either expression vector. This facilitates the rapid and inexpensive preparation of vectors that can be used to select the RNA Pol III promoter that is best suited for your experimental protocol.

Using the pSilencer vectors, Ambion scientists have reduced the expression of GAPDH, cyclophilin, c-myc, p53, and cdk-2 (RNA and protein levels). An example experiment is shown in Figure 2. As expected, the two pSilencer plasmids are not equally effective at reducing target gene expression (Figure 2). This is likely due to differential siRNA expression levels or siRNA localization in the cells that we tested.

Figure 2. pSilencer-Induced Reductions in Target Gene Expression. pSilencer 2.0-U6 and 3.0-H1 vectors encoding hairpin siRNAs specific to GAPDH, cyclophilin, or a non-genomic sequence were transfected into HeLa cells. Forty-eight hours post-transfection, target RNA and protein levels were assessed. (A) 1 ug of total RNA isolated from various cell samples was assessed by Northern analysis using the NorthernMax™ procedure (Ambion). RNA probes specific to GAPDH, cyclophilin, and 28S rRNA were used to probe the Northerns. The specific activity of the 28S rRNA probe was approximately 100,000-fold lower than the mRNA-specific probes. (B) GAPDH protein levels in cells transfected with pSilencer 2.0-U6-GAPDH were analyzed by immunofluorescence using a GAPDH-specific antibody (Ambion). Green: anti-GAPDH antibody detected with fluorescein labeled secondary antibody; Blue: DAPI stained nuclei.

The pSilencer 2.0-U6 and 3.0-H1 vectors each include four components: (1) a linearized and purified vector ready for ligation; (2) a GAPDH-specific siRNA insert that can be used as a positive control; (3) a circular, negative control pSilencer vector that expresses an siRNA with limited homology to any known sequences in the human, mouse, and rat genomes; and (4) 1X DNA Annealing Solution to prepare DNA oligonucleotides for ligation. The pSilencer 1.0-U6 vector is available in both linear and circular formats. The linear pSilencer 1.0-U6 vector is supplied with linearized and purified vector ready for ligation, and a small amount of circular vector containing a GAPDH-specific siRNA insert for use as a positive control.

REFERENCES

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

2. Tuschl T. (2002) Expanding small RNA interference. Nature Biotechnology 20, 446-448.

3. Elbashir, SM, Lendeckel, W, and Tuschl, T. (2001b) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes and Dev. 15, 188-200.

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, 5515-5520.

5. Myslinski E, Ame J-C, Krol A, Carbon P (2001) An Unusually Compact External Promoter for RNA Polymerase III Transcription of the Human H1 RNA Gene. Nucl. Acids, Res. 29, 2502-09.

6. Kunkel GR and Pederson T (1989) Transcription of a human U6 small nuclear RNA gene in vivo withstands deletion of intragenic sequences but not of an upstream TATATA box. Nucl. Acids Res. 17, 7371-7379.

7. Ilves H, Barske C, Junker U, Bohnlein E, Veres G (1996) Retroviral vectors designed for targeted expression of RNA-Polymerase III-Driven Transcripts: A Comparative Study. Gene 171, 203-208.

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

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