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A System for Stable Expression of Short Interfering RNAs in Mammalian Cells

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Science  19 Apr 2002:
Vol. 296, Issue 5567, pp. 550-553
DOI: 10.1126/science.1068999

Abstract

Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells. We show that siRNA expression mediated by this vector causes efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Stable expression of siRNAs using this vector mediates persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time. Therefore, the pSUPER vector constitutes a new and powerful system to analyze gene function in a variety of mammalian cell types.

In several organisms, introduction of double-stranded RNA has proven to be a powerful tool to suppress gene expression through a process known as RNA interference (1). However, in most mammalian cells this provokes a strong cytotoxic response (2). This non-specific effect can be circumvented by use of synthetic short [21- to 22-nucleotide(nt) interfering RNAs (siRNAs)], which can mediate strong and specific suppression of gene expression (3). However, this reduction in gene expression is transient, which severely restricts its applications. To overcome this limitation, we designed a mammalian expression vector that directs the synthesis of siRNA-like transcripts [pSUPER, suppression of endogenous RNA, Fig. 1A and Supplementary fig. 1C (4)]. We used the polymerase-III H1-RNA gene promoter, as it produces a small RNA transcript lacking a poly-adenosine tail and has a well-defined start of transcription and a termination signal consisting of five thymidines in a row (T5). Most important, the cleavage of the transcript at the termination site is after the second uridine (5) yielding a transcript resembling the ends of synthetic siRNAs, which also contain two 3' overhanging T or U nucleotides (nt) (Fig. 1A). We designed the gene-specific insert such that it specifies a 19-nt sequence derived from the target transcript, separated by a short spacer from the reverse complement of the same 19-nt sequence. The resulting transcript is predicted to fold back on itself to form a 19–base pair stem-loop structure, resembling that of C. elegans Let-7 (Fig. 1A).

Figure 1

A vector-based suppression of gene expression in mammalian cells. (A) Schematic drawing of the pSUPER vector. The H1-RNA promoter was cloned in front of the gene specific targeting sequence (19-nt sequences from the target transcript separated by a short spacer from the reverse complement of the same sequence) and five thymidines (T5) as termination signal. The predicted secondary structures of pSUPER-CDH1 transcripts and the synthetic siRNA used to target CDH1 are depicted. (B) MCF-7 cells were transfected using an electroporation protocol, which results in more than 90% transfection efficiency (7). The indicated DNA constructs (1 μg) and synthetic siRNA (1.5 μg) were transfected. Sixty hours later whole-cell extracts were prepared, separated on 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted to detect CDH1 protein. An immunoblot with antibody against cyclin D1 was used as a control. (C) Cells were transfected as described above, and total RNA was extracted 60 hours later. RNA (30 μg) was loaded on an 11% denaturing polyacrylamide gel, separated, and blotted as described (8). Membranes were probed with either32P-labeled sense or antisense 19-nt Cdh1 target oligonucleotide and visualized by PhosphorImager (overnight exposure). The stem-loop precursor and the siRNAs are indicated. The control 5S-rRNA band was detected with ethidium bromide staining. (D) Left, Cells were transfected with increasing amounts pSUPER-p53, which is predicted to produce the depicted transcript. Sixty hours after transfection cells were either irradiated (+IR, 20 Gy) or left untreated, harvested 2 hours later, and separated on 10% SDS-PAGE. Immunoblot with p53-specific antibody was preformed and the bands corresponding to p53 protein and a loading control are indicated. Middle, Northern blot showing levels of p53 mRNA in cells transfected with 1 μg of pSUPER constructs as indicated. Ethidium bromide staining of rRNAs is used as loading control. Right, Flow cytometry was performed on cells transfected with 1 μg plasmids 24 hours after irradiation as described (7). Cells with DNA in the G1 phase are indicated with an arrow.

We used the pSUPER vector to suppress the endogenous CDH1gene, an activator of the anaphase-promoting complex (APC). We designed three related vectors directing the synthesis of the same 19–base pair double-stranded CDH1 target sequence, containing loops of seven, nine, or five nucleotides (Fig. 1A, constructs A, B, and C, respectively). We compared the ability of these vectors to inhibitCDH1 to that of synthetic siRNA oligonucleotides targeted against the same sequence of CDH1 (Fig. 1A) in a transient expression experiment in MCF-7 cells. Introduction of CDH1synthetic siRNA resulted in a reproducible reduction of more than 90% of CDH1 protein (Fig. 1B, lane 2). Importantly, pSUPER-CDH1-B was able to knockdown CDH1 expression to the same extent as was seen with the synthetic CDH1 siRNA (Fig. 1B, lane 5). pSUPER-CDH1-A only had a moderate activity, whereas pSUPER-CDH1-C was inactive in this assay, indicating that the size and nucleotide sequence of the loop is very important. Neither the transfection of the synthetic CDH1 siRNA, nor introduction of the siRNA expression vector, had any detrimental effect on cell survival or cell-cycle profile (6).

Next, we examined whether pSUPER vectors produce siRNA. Northern blot analysis revealed that cells transfected with pSUPER-CDH1-B produce both sense and antisense 21- to 22-nt CDH1 RNAs, which are similar in size to those seen in cells transfected with the synthetic siRNA (Fig. 1C). The 49-nt precursor transcript can be barely detected with both probes, suggesting rapid cleavage to siRNA. (Owing to its strong secondary structure, the precursor transcript migrates around 40-nt.) These results indicate that the stem-loop precursor transcript was generated and cleaved in the cell to produce a functional siRNA.

To examine the specificity of our pSUPER system, we constructed two CDH1 vectors harboring a mutation at either position 2 or 9 of theCDH1 target recognition sequence (Fig. 2A). Transient transfection of these constructs into cells failed to suppress CDH1 expression, although both vectors yielded transcripts of the length of siRNAs [Fig. 2A and (6)]. These results demonstrate that suppression of gene expression by pSUPER system is highly target sequence-specific.

Figure 2

(A) An intact target recognition sequence is required to suppress CDH1 by pSUPER-CDH1 vector. The CDH119-nt target-recognition sequence was mutated to give a 1–base pair substitution at either position 9 or 2 of the stem. The predicted secondary structures of the transcripts are shown. U2OS cells were transfected exactly as described in Fig. 1. Whole-cell extracts were prepared after 60 hours, separated on 10% SDS-PAGE, and analyzed by immunoblotting with CDH1-specific antibody. Cyclin D1 protein was used to demonstrate equal loading. (B) Suppression ofCDC20 expression by both synthetic siRNA and pSUPER-CDC20. Shown are the sequences of the siRNA and the predicted transcript of pSUPER-CDC20 utilized to knockdown CDC20 expression. The indicated siRNAs and plasmids were transfected into MCF-7 cells as described above. Whole-cell extracts were separated on 10% SDS-PAGE and immunoblotted to detect CDC20 and cyclin D1 proteins. Two transfections from each vector are shown.

To further test the vector system, we designed both a synthetic siRNA and a pSUPER vector that target the same 19-nt sequence in theCDC20 transcript. As for CDH1, efficient suppression of endogenous CDC20 expression was achieved with both synthetic siRNA and with pSUPER-CDC20 (Fig. 2B). To measure the level of gene suppression accurately by the pSUPER system, we designed a construct to target polo like kinase-1 (PLK1). Introduction of pSUPER-PLK1 led to a significant decrease in PLK1 protein levels and a reduction in PLK1 kinase activity by a factor of 10 [Supplementary fig. 1A (4)]. To date, we were successful in knocking down the expression of more than 10 genes for which we designed a pSUPER siRNA vector, highlighting the efficiency with which genes can be targeted using this vector (6).

Next, we asked whether suppression of gene expression by the pSUPER vector is sufficient to affect cellular physiology. We designed a construct to knockdown p53, a transcription factor that is stabilized following ionizing radiation (IR) and plays a crucial role in the maintenance of cell-cycle arrest in G1 after DNA damage (7). Transfection of pSUPER-p53 reduced endogenous and overexpressed p53 protein [Fig. 1D, left, and Supplementary fig. 1B (4)] to very low levels and prevented entirely its induction after IR (Fig. 1D, left). Suppression of p53 was at the mRNA level, which was reduced by at least 90%, as judged by PhosphorImager quantification (Fig. 1D, middle). When vector-transfected cells were irradiated, they arrested within 24 hours in either G1 or G2 with very few cells remaining in S phase (Fig. 1D, right). In contrast, cells transfected with the pSUPER-p53 almost completely lost their p53-dependent arrest in G1 but were able to establish a p53-independent G2/M arrest. These results indicate that our vector can suppress endogenous p53 expression to the extent that it completely abrogates its function in the DNA damage response.

Finally, we asked whether the pSUPER vector can mediate stable suppression of gene expression. MCF-7 cells were co-transfected with a vector containing the puromycin-resistance marker and either pSUPER or pSUPER-p53. Cells were selected with puromycin, and resistant clones were cultured. When analyzed after 2 months, all pSUPER-transfected control clones stained brightly with p53-specific antibody, whereas more than 50% of pSUPER-p53 transfected clones showed significant reduction in p53 level (Fig. 3, A and B). In these stable clones p53-specific siRNA production was clearly detected (Fig. 3C, lanes 2 and 3) and was comparable to the level obtained after transient transfection of pSUPER-p53 (lane 1). These results indicate that the knockdown mediated by pSUPER is maintained over long periods and that its transcript products are not toxic to cells, as no selection against p53 knockdown was observed.

Figure 3

Stable suppression of gene expression. Cells transfected with 1 μg of pSUPER vectors and 0.1 μg of pBabe-puro plasmid were selected with 1 μg/ml puromycin for 12 days. Clones were picked and expanded for an additional 2 months and analyzed for p53 protein levels. (A) Immunofluorescence using antibodies against p53 (green) and against actin, as a control (red). (B) Immunoblot analysis for p53 and control (CDK4). (C) Stable clones for pSUPER and pSUPER-p53 after 2 months in culture (lanes 2 and 3) and transiently transfected cells with 1 μg pSUPER-p53 after 48 hours (lane 1) were analyzed for p53-specific siRNAs expression as described in Fig. 1C. Blots were probed with a 32P-labeled sense p53 19-nt probe corresponding to the targeting sequence.

In summary, we provide a powerful new tool to stably suppress gene expression in mammalian cells, which will be useful in a variety of biological systems. Our finding that a single nucleotide mismatch in the 19-nt targeting sequence abrogates the ability to suppress gene expression also opens new avenues for gene therapy. Vectors that target disease-derived transcripts with point mutations, such as those from mutant RAS or TP53 oncogenes, can now be specifically designed, without altering the expression of the remaining wild-type allele. In addition it should be possible to generate large collections of pSUPER siRNA vectors to carry out high-throughput genetic screens for loss-of-function phenotypes.

  • * To whom correspondence should be addressed. E-mail: r.agami{at}nki.nl

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