A plasmid-based system for expressing small interfering RNA libraries in mammalian cells
© Kaykas and Moon; licensee BioMed Central Ltd. 2004
Received: 04 February 2004
Accepted: 30 April 2004
Published: 30 April 2004
RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21–25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence.
To circumvent the limitation of hairpin siRNA vectors we have developed a convergent opposing siRNA expression system called pHippy. We have generated pHippy vectors or expression cassettes that knock down the expression of both reporter and endogenous genes. As a proof of principle that pHippy can be used to generate random siRNA libraries, we generated a small siRNA library against PGL3 luciferase and demonstrated that we could recover functional siRNAs that knock down PGL3 luciferase.
siRNA is a powerful tool to study gene function. We have developed a new vector with opposing convergent promoters for the expression of siRNAs, which can be used to knock down endogenous genes in a high throughput manner or to perform functional screening with random or cDNA-derived siRNA libraries.
RNA interference is an evolutionarily conserved process that functions to inhibit gene expression [1, 2]. The phenomenon of RNAi was first described by Fire et al. six years ago. In seminal experiments they injected double stranded RNA (dsRNA) into Caenorhabditis elegans, which led to efficient sequence-specific gene silencing of the mRNA that was complimentary to the dsRNA . RNAi has also been described in plants as a phenomenon called post-transcriptional gene silencing (PTGS) which is likely used as a viral defense mechanism [4–6]. Introduction of long dsRNA into a variety of organisms such as Drosophila, Trypanosoma, and pre-implanted mouse oocytes has been shown to specifically inhibit the complimentary mRNA [5–7]. However, in somatic mammalian cells long dsRNA also induces the interferon response, which globally inhibits translation by induction of the kinase, PKR, and 2', 5'-oligoadenylate synthetase. This has limited the use of RNAi as a tool to study gene function in mammalian cells [8, 9].
The first indication that the molecules that regulate PTGS were short RNAs processed from longer dsRNA was the identification of short 21 nt to 22 nt dsRNA derived from the longer dsRNA in plants . This observation was recapitulated in Drosophila embryo extracts where long dsRNA was found processed into 21–25 nt short RNA by the RNase III type enzyme, Dicer [9–11]. These observations led Elbashir et al. to test if synthetic 21–25 nt synthetic dsRNAs function to specifically inhibit gene expression in Drosophila embryo lysates and mammalian cell culture [9–11]. They demonstrated that siRNA had the ability to specifically inhibit gene expression in mammalian cell culture without induction of the interferon response. These observations led to the development of many techniques for the specific knockdown of genes in mammalian cell culture.
Of these techniques, plasmid-based systems that generate hairpin siRNAs are very appealing [12–15]. These vectors are fairly inexpensive and have been shown to inhibit multiple genes both transiently and in long-term experiments. However, hairpin vectors suffer from multiple limitations. Hairpins can be hard to synthesize in bacteria, difficult to sequence, and the oligos needed to generate them can be costly and error-prone [14, 16]. In addition, the hairpin length and sequence can affect the ability of the siRNA to inhibit gene expression [12, 17]. Although cDNA-based siRNA libraries have been generated in hairpin vectors, generation of these libraries is technically difficult and heterologous hairpin loops have to be added to the siRNA [18, 19]. The latter may increase off-target effects of the siRNAs generated from these strategies.
To circumvent the limitations of hairpin siRNA expression vectors, we have generated a siRNA expression vector, which expresses siRNAs from convergent opposing promoters. We have named this vector "HI i nverted U6 p romoter p lasmid," or pHippy. Short double-stranded oligonucleotides can be easily and efficiently introduced into this vector to knock down any mRNA, or siRNA expression cassettes can be generated in a high throughput manner by PCR. We demonstrate that pHippy can generate functional siRNAs in mammalian cells that knock down the expression of both ectopic reporters and endogenous genes. Because both strands of the siRNA are transcribed from the same template DNA, random or cDNA-derived siRNA libraries can easily be generated in pHippy. As a proof of principle we generated a random pool of siRNAs against PGL3 luciferase and screened this library for silencing activity. This library was deconvoluted to identify functional vectors that have the original siRNA sequence against PGL3 luciferase. Although this is only a partial random library to a previous efficacious siRNA, this demonstrates that random siRNA libraries can be used to functionally identify genes.
Results and discussion
Design of an opposing convergent siRNA expression vector, pHippy
pHippy efficiently inhibits the ectopic expression of reporter genes
To determine if pHippy generates sequence-specific inhibitory siRNAs, a vector was designed against EGFP. pHippyEGFP did not inhibit the expression of PGL3 Luciferase (Fig. 2a). However, it inhibited the expression of EGFP (Fig. 2b). This not only establishes that the inhibition of expression of EGFP and Luciferase is gene-specific, but also demonstrates that pHippy can theoretically be used to knock down the expression of any gene. Supportingly, to date we have knocked down expression of more than 10 unique genes with pHippy (data not shown). We have not observed off-target effect of these siRNAs, though we have not systematically screened for such potential effects. We speculate that siRNAs generated by pHippy are less likely to generate off-target effects because there are no heterologous sequences included in the siRNA, such as a hairpin loop. On average we have found that 3–4 constructs with unique target sequences have to be tested to obtain functional siRNA, which is very similar to other siRNA systems.
pHippy efficiently inhibits the expression of endogenous genes
pHippy expression cassettes can easily be generated in a high throughput manner by PCR
Generation of random siRNA-libraries in pHippy
These experiments demonstrate that the pHippy system can be used for random siRNA screens. Specifically, libraries can be generated where all of the 21 nt of the siRNA are random. This library would encompass multiple targets in every gene in the human genome and could be used for phenotypic single cell assays to identify genes required for the screened phenotype, without first knowing the siRNA sequence. For instance, a random siRNA library could be used to identify genes required for Wnt signaling by screening for siRNAs that inhibit the ability of Wnt to activate Super(8X)Topflash.
pHippy is an siRNA expression vector, which utilizes convergent promoters to generate functional siRNAs by driving expression from both strands of the same template DNA. In tests to date, pHippy vectors directed against PGL3 luciferase knock down the expression of luciferase better than hairpin RNAs. This is likely because pHippy produces functional siRNA that do not need to be cleaved by Dicer. In addition, pHippy vectors against any target gene are easier to generate than with hairpin vectors. Specifically, the oligos needed to generate pHippy siRNA vectors are shorter and more cost effective, and the final clones are easier to identify and sequence. pHippy expression cassettes can be generated in a high throughput manner by PCR without the need to propagate through bacteria.
We have used pHippy to knock out the expression of an endogenous Wnt pathway co-receptor, as scored by measuring effects on a Wnt pathway reporter, thereby validating this approach for endogenous targets. siRNAs synthesized from pHippy are predicted to have fewer off-target effects than some siRNA approaches since pHippy-derived siRNAs have no extraneous nucleotides, a feature one might design into vectors developed with gene therapy as a goal.
One of the biggest advantages is that pHippy can be used to generate random and cDNA siRNA expression libraries, which can be used to identify genes involved in any biological phenomena that can be screened by phenotype. As a proof of principle we screened a small random siRNA library for siRNAs that inhibit PGL3 luciferase activity, and were able to recover functional siRNAs that knocked down activity of this target. This approach can now be used to identify siRNAs that modulate diverse biological phenomena.
Cell culture and transfection
293T cells were grown in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin under standard conditions. All transfections were performed in 24 well plates with Lipofectamine Plus or 2000 (Invitrogen) according to the manufacturer's specifications.
2.5. × l05 293T cells were seeded in 12 well plates and transfected with 10 ng of pEGFPNl (Clontech), pDSREDNl (Clontech), and 30 ng of the indicated siRNA construct. 24 hours after transfection the cells were removed from the plates with PBS and live cells were sandwiched between cover-slips and glass slides and visualized for fluorescence using the appropriate lasers and filters to visualize EGFP and DSRED.
Luciferase assays were preformed according to the Dual luciferase assay specifications (Promega). In all cases 293T cells were transfected with 10 ng of CMV-PGL31uciferase and 100 pg of pRLCMV (Promega), and the cells were harvested 24 hours later and assayed for luciferase activity in 96 well plate in a Berthold 96 V luminometer. Super(8X)Topflash reporter assays were performed as described . 293T cells seeded in 24 well plates were transfected with 10 ng of Super(8X)Topflash, 100 pg of pRLCMV (Promega), and the indicated amount of effecter plasmids. The concentrations of all transfections were brought up to a total of 250 ng with the vector CS2+. Assays were performed as described  and according to the Dual luciferase assay specifications (Promega).
Multiple primer PCR
pHippy siRNA expression cassettes were generated by a single step multiple primer PCR. In short, a 10 ng of plasmid containing the human U6 promoter was used as template for PCR in a 50 μl reaction containing 2 μl of 10 pm/μl U6 primer, 2 μl of a primer encompassing the entire H1 promoter, 2 μl of 0.01 pm/μl of the gene-specific linker primer, 10 μl of 2 mM DNTPs, 10 μl of advantage buffer, and 0.5 μl tag-advantage (Clontech). The PCR products were generate by 30 cycles of touchdown PCR program that ramped down from 60°C to 50°C.
RNAi design and recombinant DNA constructs
All recombinant constructs were generated by standard recombinant DNA techniques. The sequences of pHippy will be provided upon request. siRNAs were designed using the web based siRNA design program from the Whitehead Institute web page http://jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/home.php. In general, siRNAs were used from previously published siRNAs or were designed by introducing the corresponding sequence for a given gene into the Whitehead siRNA design program using the following pattern AAN19TT or AAGN17CN2. The output sequences for a given gene that were not complimentary to other genes after blasting were chosen, and the corresponding oligos were designed and ordered. Oligos were based on the Whitehead output sequences and modified for cloning into pHippy by addition of 2 or 4 adenines to the 5' end of the sense and antisense versions of the Whitehead output sequences. Oligos used; PGL3luciferase, sense (5'AAaaggctcctcagaaacagctc3'), antisepses (5'AAaagagctgtttctgaggagcc3'): EGFP, sense (5' AAaagcaagctgaccctgaagttcat3'), antisense (5' AAaaatgaacttcagggtcagcttgc3'): LRP6#1 sense (5'AAaaaggttcccttccacatcct3'), antisense (5'AAaaaggatgtggaagggaacct3'): LRP6#2 sense (5'AAaaaaggttcccttccacatccttt3'), antisense (5'AAaaaaggatgtggaagggaaccttt3'): LRP6#3 sense (5'AAaagaagatggcagccagggct3'), antisense (5'AAaaagccctggctgccatcttc3'): LRP6#4 sense (5'AAaaggcacttacttccctgcaa3'), antisense (5'AAaattgcagggaagtaagtgcc3'): LRP6#5 sense (5'AAaaaaggcacttacttccctgcaatt3'), antisense (5'AAaaaattgcagggaagtaagtgcctt3').
We thank Bryan White and Stephane Angers for comments on the manuscript. AK is an Associate, and RTM an Investigator, of the HHMI, which supported this work.
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