To verify that the GFP expression level could be evaluated by flow cytometry, we first examined siRNA directly targeted to GFP. An MSCV-based vector (MSCV-IRES-GFP: pMIG) was utilized as our GFP expression vector. An siRNA sequence against GFP was cloned into the siVM2 expression vector, in which siRNA was driven by a human H1 promoter (siVM2GFP) [19
]. A red RFP monomer expression vector (pDsRed-Momoner-Hyg-N1: pRFP) was used to normalize the transduction efficiency.
HEK293 cells were transduced with pRFP and pMIG with or without siVM2GFP, and the GFP and RFP expression levels were evaluated by fluorescent microscopy and flow cytometry 24 h after transfection (, respectively). When the RFP expression vector was co-transduced with GFP, most RFP-positive cells expressed GFP, as expected (middle panels in ). The GFP expression was highly suppressed when siVM2GFP was co-transduced (bottom panels in ). In contrast, the RFP expression level was not affected by addition of the GFP-targeted siRNA expression vector, suggesting that the use of RFP is appropriate for correcting the transduction efficiency.
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 (more ...)
displays the mean fluorescent intensities (MFIs) for GFP within RFP-positive cells. The solid and dotted lines represent GFP expression with or without the GFP expression vector, respectively, while the shaded line represents GFP expression in RFP-positive cells transduced with the siRNA expression vector. The MFIs for each condition were: 5.06 for pRFP only, 226 for pMIG + pRFP, and 37.5 for siVM2GFP + pMIG + pRFP. The siRNA efficiency was calculated as 85.3%, according to the following equation: siRNA efficiency = (MFIpMIG+pRFP − MFIpMIG+pRFP+siVM2) / (MFIpMIG+pRFP − MFIpRFPonly). These results suggest that the GFP expression level within RFP-positive cells could be examined at the single cell level by flow cytometry.
Using this system, we assessed three different siRNA sequences targeting the mouse CREM protein. CREM is a member of the cyclic AMP responsive element binding (CREB) protein family of transcription factors. The siRNAs were designed to target the mouse CREM (NM_013498) coding sequence at 308-, 469-, and 817-bp. As a non-specific siRNA control, we used siRNA targeted to firefly luciferase (GL3, Promega). shows the GFP and RFP expression levels in cells transduced with a GFP expression vector without mouse CREM sequences (pMIG). None of the siRNAs affected the GFP or RFP expression levels (, left panel). The solid and dotted lines in (right panels) represent the GFP expression in RFP-positive cells with or without pMIG, respectively. The shaded lines represent the GFP expression levels with transduction of the siRNA expression vectors. The GFP expression within RFP-positive cells was not repressed by any siRNA vector.
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], (more ...)
Next, HEK293 cells were transduced with pRFP and a bicistronic CREM expression vector (pMIG-mCREM), with or without an siRNA expression vector. CREM-targeted, but not luciferase-targeted, siRNAs specifically repressed GFP expression (, left panels). The GFP expression levels of RFP-positive cells are plotted in the right panels of . The solid and dotted lines represent GFP expression within RFP-positive cells transduced with or without pMIG-mCREM, respectively. The shaded lines represent GFP expression with transduction of the siRNA expression vectors. The MFIs for each condition were as follows: 6.19 for pRFP only, 59 for pMIG-mCREM + pRFP, 46.4 for pMIG-mCREM + pRFP + siVM2GL3, 9.59 for pMIG-mCREM + pRFP + siVM2CREM#1, 8.88 for pMIG-mCREM + pRFP + siVM2CREM#2, and 14.8 for pMIG-mCREM + pRFP + siVM2CREM#3 (, right panels). All three siRNA sequences targeting mouse CREM (CREM#1, #2, and #3) effectively suppressed GFP expression in RFP-positive cells compared to the control (no siRNA expression vector), with efficiencies of 93.5%, 94.9%, and 83.6%, respectively. The siRNA for firefly luciferase did not affect GFP expression. These results suggest that the effects of the siRNA vectors on the GFP expression level were specific to the CREM sequence located in front of the IRES sequence, and not to the GFP sequence.
To verify our screening method, we measured the mRNA and protein expression levels using real time PCR and Western blotting, respectively (). The total RNAs and proteins for the measurement were extracted from the same samples as used in the flow cytometric analysis. Both the mRNA and protein expression levels of CREM correlated very well with the relative MFI for GFP in RFP-positive cells (, R2 = 0.9537 and 0.9171, respectively). We also verified the feasibility of the system by applying it to four other genes (data not shown).
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], (more ...)