A plasmid-based system for expressing small interfering RNA libraries in mammalian cells

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Depiction of the pHippy dual siRNA expression vector and some of its possible uses. a, pHippy has convergent opposing Human H1 and U6 polymerase III promoters that drive expression of both strands of any template cloned in between the BsmB 1 cloning sites. pHippy also contains the Puc origin and the Zeocin-resistance gene for propagation and replication in bacteria. As depicted the H1 and U6 promoters were modified to contain a polymerase III termination signal (TTTTT) between the -5 to -1 position of the promoter, and BsmBl sites. BsmBl is a type II restriction enzyme, which cuts outside of its recognition sequence and will in the case of pHippy leaves 3' TTTT overhangs on both strands of the plasmid as depicted. 19 nt target siRNA can be cloned into pHippy as double stranded oligos by addition of AAAA to the 5' ends of the oligos as depicted b. b, Proposed uses for the pHippy siRNA vector system.

Figure 2

a, Luciferase assay to determine the efficiency of target suppression by the pHippy vector. Hairpin siRNA expression vectors were generated against PGL3 luciferase with either the U6 (U6GPL31ucHP) or H1 (HlPGL31ucHP) promoters. For comparison, pHippy siRNA vectors were generated against PGL3 luciferase (pHippyPGL31uc) or against EGFP (pHippyEGFP). 293T cells were transfected with the constructs shown and assayed for luciferase activity 24 hours later. The absolute levels of PGL3 luciferase were ~100,000 relative light units (RLUs) which was set at 100%. All experiments were normalized for transfection efficiency with an expression vector for Renilla luciferase. The average normalized PGL3 luciferase levels and standard deviation are shown for 3 experiments. b, EGFP fluorescent assay to monitor the suppression of protein expression by pHippyEGFP. 293T cells were co-transfected with empty pHippy or pHippyEGFP and expression vectors for DsRED and EGFP. Confocal image of EGFP and EGFP merged with DsRED are shown. One representative experiment is shown.

Figure 3

Assays to determine if pHippy can be used to inhibit endogenous genes. a, Renilla luciferase assays to determine if any of 5 unique pHippy constructs against Human LRP6 inhibit activity of a fusion of LRP6 to Renilla luciferase. 293T were co-transfected with the vectors shown and assayed for luciferase activity 48 hours later. Cells co-transfected with LRP6Rluc and empty pHippy were set to 100%. All experiments were normalized for transfection with PGL3 luciferase. The average normalized Renilla luciferase levels and standard deviation are shown for 3 experiments. b, Determination of whether inhibition of endogenous LRP6 inhibits Wnt3a activation of the Wnt/β-catenin signaling pathway. 293T cells were co-transfected with the vectors shown, Super(8X)Topflash (to measure Wnt/β-catenin-signaling), and a constitutive expression vector for Renilla luciferase. 24 hours after transfection the cells were treated with Wnt3a conditioned media for 24 hours and then assayed for luciferase activity. Untreated cells transfected with empty vector were set to 1 fold activation of Super(8X)Topflash, which corresponds to ~10,000 RLUs. Cells treated with Wnt3a induce about 100 fold-activation of luciferase as shown. All experiments were normalized for transfection efficiency with the Renilla luciferase expression vector. One representative experiment is shown.

Figure 4

A high throughput system to generate pHippy expression cassettes. a, A multiple primer PCR system was used to generate PCR products that express siRNA after transfection into cells. A 5' primer (U6 primer) was designed to human U6. A second primer (gene specific) was designed with sequences that are complementary to the 3' end of the human 6 promoter, the target siRNA sequence, and a sequence that is complementary to the 3' of the human H1 promoter. A third primer was designed that contains the human H1 promoter and is complementary to the gene specific primer. b, An agarose gel stained with ethidium bromide demonstrates the robustness of the PCR method for several different gene specific primers. c, A luciferase assay as described in the legend to Figure 3b to determine if the PCR products inhibit PGL3 luciferase activity.

Figure 5

A siRNA screen was conducted with a small siRNA library against PGL3 luciferase. a, Depiction of the small siRNA library against PGL3 luciferase. The target sequence for the siRNA against PGL3 siRNA that was tested in Figure 3b is shown. The bases in bold were randomized by synthesizing an oligo with random bases in place of CTC and cloned into the pHippy vector. b, Screening of the siRNA library. 12 pools of 10 bacterial colonies from the random siRNA library against PGL3 luciferase were screened for their ability to inhibit PGL3 luciferase activity as described in the legend to Figure 3b. c, The sequences of the siRNA single clones, which did or did not inhibit PGL3 luciferase. Pools 8 and 11 were broken down into individual clones and re-screened for inhibitory activity. Pool 8 contained 1 positive clone and Pool 11 contained 2 positive clones. The sequences of all three positive clones is shown on the left and corresponds to the original PGL3 siRNA. The sequences of 10 negative clones are shown on the right.