RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Abstract

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.

Figure 1

Strategy to generate T7 siRNAs (see Results and Discussion for details). The sequence of the gene of interest is shown in red (sense) or blue (antisense), while the two unrelated nucleotides are in black. RISC stands for the RNA-induced silencing complex that targets the mRNA for cleavage.

Figure 2

T7 siRNAs silence transfected genes in mammalian cells. (A) Plasmid pEGFP-C1 and decreasing amounts of siRNAs were co-transfected using a lipofectamine-based protocol. GFP was revealed with an anti-GFP antibody. T7 siRNA against PKR was transfected as a specificity control. (B) T7 siRNAs directed against GFP (1–3 represent siRNAs from three independent transcription reactions) or PKR were transfected using the calcium phosphate co-precipitation technique into HeLa cells with the plasmids pEGFP-C1 and pcDNA3-Luc. GFP and luciferase were detected with anti-GFP and anti-luciferase polyclonal antibodies, respectively.

Figure 3

T7 siRNA silences an endogenous gene in mammalian cells. Aliquots of 0.2 or 2 µl of T7 siRNA directed against PKR were transfected using the calcium phosphate co-precipitation technique into 293T cells. Forty hours after transfection, cells were harvested and equal amounts of proteins were loaded on a 10% polyacrylamide gel. PKR was detected using a polyclonal antibody against PKR. Hsp90 was revealed as a specificity and loading control.