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. 2003 Nov 6:3:21.
doi: 10.1186/1472-6750-3-21.

Expressing functional siRNAs in mammalian cells using convergent transcription

Nham Tran  1 , Murray J Cairns, Ian W Dawes, Greg M Arndt
Affiliations

Expressing functional siRNAs in mammalian cells using convergent transcription

Nham Tran et al. BMC Biotechnol. .

Abstract

Background: The use of small interfering RNAs (siRNAs) as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs) or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi)-mediated gene silencing.

Results: With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells.

Conclusion: The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences. Furthermore, the cassette design reported is ideal for both routine use in controlling specific gene expression and construction of randomised RNAi expression libraries for use in unbiased forward genetic selections.

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Figures

Figure 1
Figure 1
Strategy for generating intracellular siRNAs and effect of the expressed siRNAs on transgene expression. (A) The convergent U6 expression cassette encodes sense (black) and antisense RNAs (red) that terminate at directional termination sequences (represented as five consecutive thymidines [T]). The complementary RNAs anneal and undergo further Dicer-dependent processing to produce functional siRNAs. A U6 convergent expression vector containing an EGFP-specific insert (DualU6GFP) reduces dEGFP-mediated cell fluorescence as measured by (B) flow cytometry and (C) fluorescence microscopy. This same expression vector suppresses both (D) dEGFP protein levels and (E) dEGFP RNA levels. For Western analysis, β-actin protein levels were used as a loading control. For RNA analysis, the control for loading was 18s rRNA.
Figure 2
Figure 2
Gene suppression by complementary RNAs expressed from a U6 convergent cassette is Dicer-dependent. (A) The U6 convergent EGFP vector undergoes transcription in EcR293 cells to produce sense and antisense RNAs. (B) Suppression of target gene expression by the DualU6GFP vector requires the co-expression of both sense and antisense RNAs. (C) The DualU6GFP expression vector reduces dEGFP target gene expression in a Dicer-dependent manner. (D) Co-expression of complementary RNAs from the U6 convergent expression cassette does not activate PKR. Hela cells were treated with 0.1 μM calyculin A and serve as a positive control for activated PKR.
Figure 3
Figure 3
Stable suppression of dEGFP-mediated cell fluorescence. EcR293 cells containing a stable integrated dEGFP transgene were co-transfected with DualU6GFP and pREP7 and selected in 500 ug/ml hygromycin for two weeks. Cell fluorescence is reduced in stable pools of cells containing the DualU6GFP vector compared with those receiving the control DualU6 vector.
Figure 4
Figure 4
Suppression of p53 protein levels using a convergent U6 expression vector. (A) Transient suppression of p53 protein levels. Plasmids DualU6 and DualU6p53 or p53-specific siRNAs 1 and 2 were transfected into MDA MB 231 cells and, at 48 h and 120 h post-transfection, p53 and β-actin protein levels were determined using Western analysis. (B) Long term suppression of p53 protein levels. EcR293 cells containing a stably integrated dEGFP transgene were co-transfected with DualU6, DualU6GFP or DualU6p53 and pREP7. Following selection in hygromycin, cell populations were analysed for p53 and β-actin protein levels.
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