A universal transgene silencing method based on RNA interference

Abstract

Inducible gene expression systems have contributed significantly to the understanding of molecular regulatory networks. Here we describe a simple and powerful RNA interference-based method that can silence the expression of any transgene. We first used an IRES bicistronic lentiviral vector and showed that targeting the second cistron with a specific siRNA resulted in silencing of both transgenes. We then inserted a siRNA minimal target sequence in the 3'-untranslated region (3'-UTR) of a transgene and showed that the cognate siRNA delivered by a lentiviral vector led to the partial silencing of the transgene. The multimerization of this siRNA target sequence led to the highly efficient silencing of four different transgenes. This new method to silence transgene expression is more versatile than existing methods of conditional inactivation of gene expression, such as transcriptional switches or site-specific recombination. It is applicable to a wide variety of models including primary cells, terminally differentiated cells and transgenic animals.

Figure 1

A siRNA directed against the second transgene of a bicistronic vector silences the expression of both transgenes. (A) Schematic representation of integrated proviral SIV vectors. (B) Silencing of GFP. 2 × 10⁵ HeLa cells were transduced with pSIV-TRX-i-GFP (MOI 15: 100% of transduction) and 24 h later, with two doses of SirGFP or a control construct SirGFPμ. RT activities of all viral preparations were measured to normalize doses of SirGFPs. The RT activity ratio between SirGFPs versus pSIV-TRX-i-GFP is indicated by 1/10 and 1/1. The mean fluorescence intensity was measured by flow cytometry, 72 h after transduction of SirGFPs. Results are given as percentages of residual fluorescence intensity in the global cell population. (C) Silencing of TRX. The experiment is the same as in (B). Cell lysates were subjected to a SDS–PAGE followed by a western blot analysis with antibodies against β-tubulin and the HA epitope tag. Black boxes: U3-deleted SIN LTR, gray boxes: Expression regulators (EF1, short elongation factor 1 promoter; W, woodchuck virus post-transcriptional regulatory element; H1, RNA polymerase III H1 promoter). The Ψ RRE SA region includes SIV cis-active minimal elements essential for packaging and processing of the SIV vector RNA (11). cPPT: DNA flap (14). GFP: enhanced green fluorescent protein. TRX: human thioredoxin. SirGFP codes for a small hairpin RNA targeting GFP.

Figure 2

H-2K^k transgene silencing by an siRNA directed against a 3′-UTR minimal target sequence. (A) Schematic representation of integrated proviral SIV vectors. The gray boxes depict the GFP siRNA minimal target sequence. (B) Silencing of H-2K^k expression. 3 × 10⁵ 293T cells were plated and transduced 24 h later by RT-normalized doses of H-2K^k expression vectors. Transductions with SirGFP vectors were performed 24 h later and H-2K^k expression was measured 4 days later by flow cytometry. Gray and black lines or bars depict control and SirGFP-treated cells, respectively. Percentages of H-2K^k positive cells are indicated for each condition.

Figure 3

Transgene silencing by a universal siRNA directed against a multimerized minimal target sequence. (A) Silencing of GFPμ. 293T cells were transduced with GAEts3-GFPμ. Cells were transduced with SirGFP or SirCtl, 24 h later. We checked that shRNAs coded by SirGFP did not affect GAE-directed expression of GFPμ (data not shown). Fluorescence signals were measured 72 h later by a FITC plate reader (EnVision-Perkin Elmer). (B) Silencing of TRX in HeLa cells (see Figure 1C). (C) Silencing of ZEO^R. 293T cells were transduced at low MOI with SirGFP-Flu to obtain 25% of GFP-positive cells, 72 h after transduction. Cells were then split and transduced with normalized doses of GAE-ZEO^R and GAEts3-ZEO^R (MOI 10). Zeocin was added to the culture medium 24 h later and maintained for 15 days. The silencing of ZEO^R, resulting in cell death, was monitored by determining the percentage of GFP-positive cells (i.e. the percentage of cells transduced with SirGFP-Flu) before and after the 20 day ZEO selection.