Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

Abstract

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.

Figure 1

Strategy and experimental design. The target gene and GFP are transcribed as a single mRNA from a bicistronic vector and translated independently (left panel). An siRNA against the target gene destroys the entire mRNA, including the target gene and GFP expressed from the bicistronic vector. The GFP expression level should reflect the effects of the siRNA (right panel). RFP is expressed from an independent vector.

Figure 2

Flow cytometry–based quantification of GFP expression using GFP-targeted siRNA. HEK293 cells were transduced with an RFP expression vector, co-transduced with or without a GFP and/or an siRNA expression vector, as indicated. Fluorescence was monitored 24 h after transduction by fluorescent microscopy (A) or flow cytometry (B and C). The mean fluorescent intensities (MFI) of GFP in the RFP-positive cells were plotted in panel C. The solid and dotted lines represent GFP expression within RFP-positive cells with or without the GFP expression vector, respectively. The GFP expression levels with the siRNA expression vectors are represented by the shaded lines. Results are representative of three independent experiments.

Figure 3

Flow cytometry-based method for screening for an effective siRNA against the mouse CREM gene. (A) HEK293 cells were transduced with an RFP vector and a GFP expression vector with or without one of the siRNA expression vectors (firefly luciferase [GL3], CREM#1, CREM#2, or CREM#3). Flow cytometry data analyzed 24 h after transduction are shown in the left panels. The RFP-positive cells (left panels) were analyzed for GFP expression (right panels). The solid and dotted lines represent GFP expression within RFP-positive cells with or without pMIG, respectively. (B) HEK293 cells were transduced with the pRFP vector and a bicistronic CREM expression vector (CREM+GFP) with or without one of the siRNA expression vectors (firefly luciferase [GL3], CREM#1, CREM#2, or CREM#3). Results are representative of three independent experiments.

Figure 4

Verification of the flow cytometry-based method by conventional methods. HEK293 cells were transduced with an RFP vector and a bicistronic CREM expression vector (CREM+GFP), with or without one of the siRNA expression vectors (firefly luciferase [GL3], CREM#1, CREM#2, or CREM#3). (A) Flow cytometric analysis 24 h after transduction. Mean fluorescent intensities of GFP in RFP-positive cells. (B) Relative expression level of mRNA for the mouse CREM gene, normalized to GAPDH. (C and D) Western blotting results. The band densities in (D) were quantified using Image J software (C). (E and F) The MFI was correlated with the mRNA (R2 = 0.9537) (E) or protein expression (R2 = 0.9171) (F). Results are representative of three independent experiments.