Mol. Cells, Vol. 18, No. 1, pp. 127-130

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Construction of a Vector Generating both siRNA and a Fluorescent Reporter: a siRNA Study in Cultured Neurons

Seung Yong Yoon, Jung Eun Choi, Onyou Hwang1, Hea Nam Hong, Heuiran Lee2, Yoo Kyum Kim2, Sung-Woo Cho1, Hyun Kim3, and DongHou Kim*

Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea; 1 Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea; 2 Department of Microbiology, University of Ulsan College of Medicine, Seoul 138-736, Korea; 3 Department of Anatomy, College of Medicine, Korea University, Seoul 136-705, Korea. (Received April 6, 2004; Accepted April 21, 2004) RNA interference is an important tool for gene silenc-ing. However, its application to primary cultured cells has been limited by low transfection efficiencies. In this work we developed a vector which encodes both siRNA and red fluorescent protein. Using this vector we could markedly suppress green fluorescent protein (GFP) and bim an endogenous gene. Primary cultured cortical neurons transfected with siRNA against dou-blecortin showed that doublecortin expression was significantly inhibited in nearly all the transfected neurons. This vector identifies the transfected cells and should be useful for loss-of-gene function studies in neurons. Keywords: Fluorescent Reporter; Neuron; siRNA. Introduction RNA interference (RNAi) is a very useful tool with which to knock-down a specific gene (Hammond et al., 2001; Hutvágner and Zamore 2002) that has been widely used in mammalian cells (Caplen et al., 2001; Elbashir et al., 2001). There are two general methods of RNAi: one uses chemically synthesized 21-nt small interfering RNA (siRNA), the other vector-based siRNA. Vector-based siRNA is superior to chemically synthesized 21-nt siRNA in that it has a more prolonged effect and is relatively inexpensive (Brummelkamp et al., 2002). However, it has limitations when used to transfect primary cultured cells and some cell lines with very low transfection efficiency * To whom correspondence should be addressed. Tel: 82-2-3010-4272; Fax: 82-2-3010-4220 E-mail: dhkim@amc.seoul.kr

because the transfected cells cannot be identified. Co-transfection of a vector-based siRNA with a reporter gene such as green fluorescent protein (GFP) could solve this problem. However, cotransfection efficiency is also low in primary cultured cells including neurons so that identi-fication of all the cells transfected with the siRNA re-mains difficult (Krichevsky and Kosik, 2002). To deal with this problem we have constructed a vector which encodes both an siRNA and red fluorescent protein (RFP). We show here that this vector can efficiently suppress both endogenous and exogenous genes. Materials and Methods Construction of pU6mRFP We first double-digested pDsRed.N1 (Clontech) with NheI and BamHI and blunt end-ligated it to generate vector pDsRed.N1.MCS-dep, which has no multiclon-ing sites (MCS). Next, we double-digested pDsRed.N1.MCS-dep at its AseI and AflII sites and purified the fragment that en-codes the CMV promoter, RFP and the SV40 polyadenylation signal. We then digested pSilencer.U6.1.0 (Ambion) with SacI and blunt end-ligated it with this fragment to generate the final vector which we named pU6mRFP (Fig. 1). We confirmed that the sequences containing the pSilencer MCS were correct. We also verified that the orientation of the DsRed fragment was 5′ to 3′ and that pU6mRFP had only single ApaI and EcoRI sites. Cell culture and transfection HEK293 cells were cultured in DMEM (Invitrogen) supplemented with L-glutamine and 10% heat-inactivated fetal bovine serum. PC12 cells were cultured in RPMI 1640 medium supplemented with L-glutamine, 15% heat-inactivated horse serum and 2.5% fetal bovine serum. Primary cultures of rat cortical neurons were prepared from the brains of embryonic day 16 pups, as described previously (Cho et al.,

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Fig. 1. The construction of pU6mRFP. pDsRed.N1.MCS-dep was double-digested at its AseI and AflII sites and the fragment containing the CMV promoter, RFP and SV40 polyadenylation signal was purified. pSilencer.U6.1.0 was digested with SacI and blunt end-ligated with this fragment to generate pU6mRFP. 2002) with some modifications. Briefly, cerebral cortices were dissected in calcium- and magnesium-free Hank’s balanced salt solution and incubated in 0.125% trypsin for 10 min at 37°C. The trypsin was inactivated with Neurobasal medium (Invitro-gen) containing 20% fetal bovine serum and the cortical tissue was further dissociated by trituration with a Pasteur pipette. The resulting suspensions were diluted with Neurobasal medium supplemented with B27 components (Invitrogen) and plated onto poly-D-lysine- (Sigma, 50 µg/ml) and laminin- (Invitrogen, 1 µg/ml) coated plates. The cells were maintained at 37°C in 5% CO2 and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. siRNA sequences The siRNA sequence of the sense strand for the pEGFP reporter gene was 5′-GCAAGCTGACCCTGAAG-TTCAT-3′ as previously published (Caplen et al., 2001). The corresponding sequence for siBim was 5′-ACGAGTTCAATG-AGACTTA-3′, which was derived from the NCBI database (ac-cession number AF065433). A BLAST search showed that it had no significant sequence homology to other genes. The target sequence of siDoublecortin was 5′-GCTCAAGTGACCAAC-AAGGCTAT-3′ (3′ untranslated region of doublecortin, AF-155959) as recently published (Bai et al., 2003). 19 to 23-nucleotide sequences separated by a 9-bp hairpin loop from the reverse complementary repeats with five thymidines as termina-tion signal were synthesized by Genotech (Korea) and inserted into pU6mRFP. Immunocytochemistry Cultures were fixed with 4% parafor-maldehyde in 0.1 M phosphate buffer for 30 min, and cell mem-

branes permeabilized by incubation in PBS containing 0.1% Triton X-100 and 2% BSA for 30 min. Cultures were incubated overnight at 4°C in primary anti-doublecortin antibody solution (1:500), then washed and incubated with FITC-conjugated sec-ondary antibody (1:300, Jackson Lab). Images were acquired with a CoolSNAP CCD-camera and processed using Adobe Photoshop 6. Analysis of gene expression GFP expression was assessed by fluorescence microscopy. All pictures were taken with a CoolS-NAP CCD-camera with the same exposure time. For each con-struct, one hundred cells were examined, as previously de-scribed with minor modification (Jacobsen and Staines, 2004; Krichevsky and Kosik, 2002). In each doublecortin-targeting experiment, gene expression was quantified in the non-transfected control, and in pU6mRFP-siGFP and pU6mRFP-siDoublecortin transfected neurons. At least 100 neurons were analyzed per condition. Quantification of fluorescence intensity was per-formed with MetaVue Imaging software (Universal Imaging Cooperation). Fluorescence signal intensities were obtained by manually outlining cells for each condition and using the inte-grated densitometry function to calculate mean signal intensities for the outlined areas. Background signals were subtracted from the images. Statistical analysis The data are presented as means ± SD and were analyzed by Student’s t-test. Differences were considered statistically significant when p < 0.05. Results and Discussion To test our method, we cloned an insert encoding siRNA targeting GFP (siGFP) into pU6mRFP at the ApaI and EcoRI sites. HEK293 cells were seeded in 48-well plates and after 24 h we added 50 ng of pEGFP.N1 (Clontech) and 350 ng of pU6mRFP, or 50 ng of pEGFP.N1 and 350 ng of pU6mRFP-siGFP, to the wells (see Materials and Methods for further details). Seventy two hours after transfection, many cells gave a strong red fluorescence signal (Fig. 2, upper panel), demonstrating that RFP was efficiently expressed under the control of CMV promoter. On the other hand, the green fluorescence signal declined by 50% in the cells cotransfected with pEGFP.N1 and pU6mRFP-siGFP (Fig. 2, lower panel). This confirmed that RNA interference was occurring against GFP, but not RFP, in the cells transfected with pU6mRFP-siGFP.

To investigate whether pU6mRFP was also effective with endogenous genes we generated a construct contain-ing siRNA against Bim (pU6mRFP-siBim) (see Materi-als and Methods) and used it to transfect the PC12 cells. Other PC12 cells were transfected with pU6mRFP-siGFP as a control. Seventy two hours after transfection, the cells were collected, and Western blot analysis with Bim (Stressgen, 1:1000) and β-actin (Sigma, 1:1000) antibod-

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B Fig. 2. Effect of pU6mRFP-siGFP on HEK293 cells. HEK293 cells were cotransfected with pEGFP.N1 and pU6mRFP-siGFP (70−80% efficiency). A. Seventy two hours after the transfec-tion, there were bright red fluorescence signals (upper panel). The green fluorescence signal decreased in the cells cotrans-fected with pEGFP.N1 and pU6mRFP-siGFP (lower, right panel) but not in the cells cotransfected with pEGFP.N1 and pU6mRFP (lower, left panel) (bar = 50 µm). B. Quantification of GFP expression in RFP expressing cell after pU6mRFP-siGFP transfection. Green fluorescence was normalized to the control level (100%). At least 100 randomly chosen control (pU6mRFP) and targeted (pU-6mRFP-siGFP) cells were ana-lyzed % in each experiment. Error bars indicate SDs (p < 0.01). ies showed that Bim expression had declined by more than 60% in the cells transfected with pU6mRFP-siBim (Fig. 3). Thus, pU6mRFP-siBim can also silence an en-dogenous gene.

To assess the effectiveness of vector pU6mRFP with pri-mary cultured neurons, we transfected pU6mRFP-siDoublecortin and pU6mRFP-siGFP into IVD-5 cortical neurons. Three days after the transfection the transfected neurons had normal morphology and immunostained with doublecortin antibody (Fig. 4A), and the doublecortin was localized in the cell bodies and leading processes (Fig. 4A, a).

Fig. 3. pU6mRFP-siBim silences endogenous Bim in PC12 cells. PC12 cells were transfected with pU6mRFP-siGFP and pU6mRFP- siBim (40−50% transfection efficiency). Seventy two hours after the transfection, the cells were collected and analyzed by West-ern blotting. Data are averages of three independent experiments (see Materials and Methods for further details). However most of the neurons transfected with pU6mRFP-siDoublecortin had a high level of RFP and a considera-bly lower level of doublecortin (Fig. 4A, b and b’, arrow). To assess the efficiency of gene silencing we performed quantitative densitometry of the immunofluorescence sig-nals. This analysis demonstrated that doublecortin immu-noreactivity was reduced by 78% by the pU6mRFP-siDoublecortin relative to the siGFP-transfected neurons (Fig. 4B).

pU6mRFP encodes both siRNA and RFP. It uses the polymerase-III U6-RNA gene promoter for the siRNA and the polymerase-II CMV promoter for RFP. Because it uses distinct polymerases for the siRNA and RFP, the production of siRNA and RFP should not interfere with each other. This is demonstrated by our observation that when a specific gene was knocked-down RFP was still abundantly produced. Coexpression of RFP with silencing RNA allows identification of those cells that contain siRNA, which is essential for studying gene function in primary cultured cells such as neurons or stem cells, which have very low transfection efficiencies. The RFP reporter may also permit the production of stable cell lines with genes of choice knocked-down. In addition, the presence of the RFP makes observation of the morphol-ogy of the transfected cells easy, and this should be espe- cially useful in studying neurons, with their long proc-esses and delicate dendritic spines.

130 A Vector Generating siRNA and Fluorescent Protein �

A B Fig. 4. Knock-down of endogenous doublecortin in primary cortical neurons. A. Cultured primary cortical neurons were transfected with pU6mRFP-siGFP as control or pU6mRFP-siDoublecortin after 5 days in vitro (less than 1% transfection efficiency). After 3 days, the transfected cells were fixed and stained with doublecortin antibody (green in a, b). Fluorescence microscopy showed that the soma and processes of the siGFP-transfected neurons were doublecortin positive (a and a’, arrow), and that doublecortin expression was inhibited by transfection with pU6mRFP-siDoublecortin (b and b’, arrow). Inset shows a phase-contrast view of the region marked by the rectangle in b (bar = 20 µm). B. Quantification of doublecortin expression. At least 100 neurons were analyzed in each condition. Expression of doublecortin siRNA significantly reduced the level of expres-sion of doublecortin. Data are means ± SD (p < 0.01).

Acknowledgment This study was supported by a grant (2003-281) from the Asan Institute for Life Sciences, Seoul, Korea.

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