CHEM. RES. CHINESE UNIVERSITIES 2009, 25(5), 666—672

Construction and Characterization of Lentiviral shRNA Expression Vector Targeting Rat CD40 Gene in Dendritic Cells SUN Mei1, LI Jin-dong2, JIANG Rui3, JIN Cheng-yan2, GAO Nan4, LUO Shu-li2, WANG Chun-guang2, WANG Bin2, WANG Rong-you2 and ZHANG Xing-yi2* 1. Department of Pathology, 2. Department of Thoracic Surgery, Second Hospital of Jilin University, Changchun 130041, P. R. China; 3. Department of Orthopedic Surgery, 4. Department of Thoracic Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, P. R. China Abstract To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells(DCs), specific siRNA targets with short hairpin frame were designed and synthesized according to the mRNA sequence of rat CD40 gene. DNA oligo was cloned into lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. The verified plasmids were transfected into 293T cells that over-express recombinant CD40 in order to select the most effective siRNA targets. shRNA lentiviruses from the selected constructs were propagated and harvested with a virus packaging system, and the virus titers were determined. Western blot and Real-time PCR were performed to determine CD40 expression level in the virusinfected dendritic cells. PCR and sequencing analyses reveal that shRNA plasmids of four targets were successfully constructed. The optimal interfering target was selected, and the virus with a titer of 5×107 TU/mL was successfully packaged. CD40 expression in rat DCs was knockdown at both mRNA and protein levels by virus infection. In comparison to that of control groups, CD40 mRNA expression and protein expression were decreased by 60.9% and 61.2%, respectively. We have successfully constructed recombinant lentiviral shRNA expression vector targeting rat CD40 gene that can effectively down-regulate CD40 gene expression at mRNA and protein levels in rat DC. Keywords CD40; RNA interference; Lentivirus; Dendritic cell Article ID 1005-9040(2009)-05-666-07

1

Introduction

Immune rejection is the main cause for the failure of transplant surgery, the key to resolve the rejection is to induce or establish donor-specific immune tolerance[1―4]. It has been well documented that co-stimulatory molecules play an important role in immune response. With the development of molecular immunology, blocking co-stimulatory signals to induce immune tolerance and thereby preventing allograft rejection have become a new strategy. As CD40 is an important co-stimulatory molecule, blocking this molecule is expected to reduce host-to-donor immune response and to induce immune tolerance[2,5]. RNA interference(RNAi) is a novel technology that can efficiently and specifically down-regulate target gene and has

broad application prospects in the study of gene function and in the field of gene therapy[6―9]. However, the RNAi efficacy and transduction efficiency of small interfering RNA(siRNA) fragments are closely related to each other. Lentiviral vector can infect both dividing and non-dividing cells[10]. There is evidence to suggest that lentiviral-mediated RNAi with the advantages of stable transduction and hypo-immunogenicity is promising for gene therapy. This study is designed to construct a lentiviral-mediated RNAi vector targeting rat CD40 gene and determine its silencing efficacy in infected rat dendritic cells(DCs) in order to provide a stable transduction vector for further study on the application of blocking CD40 pathway in allograft rejection.

——————————— *Corresponding author. E-mail: [email protected] Received May 8, 2009; accepted May 31, 2009. Supported by the National Natural Science Foundation of China(No.30670301), Science and Technology Services of Jilin Province, China(No.20050408-1), PhD Scientific Research Foundation of Ministry of Education of China(No.20050183069), and the Jilin Province Talent Development Foundation(No.JRJB2007-2).

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Materials and Methods

2.1 Construction of Lentiviral Vector Expressing shRNA Four siRNA sequences targeting rat CD40(GeneBank accession No.: NM_134360) were designed with software(Ambion company) by Genechem Co., Ltd., Shanghai, China(Table 1). The siRNAs were converted into short hairpin frame(shRNA) according to Stem-Loop-Stem followed by addition of Hpa/and Xho I/restriction sites at to 5' and 3' ends. Oligonucleotides were synthesized according to the sequences and annealed into double-strands by the DNA synthesis company(Shanghai Biotechnology Co., China). Lentiviral frame plasmids were double digested and connected with the double-stranded DNA. They were then subjected to transformation of competence E. coli and positive selection of recombinant clones via PCR. PCR primers were up(+) 5'-GCCCCGGTTAATTTGCATAT-3'; down(–) GTAATACGGTTATCCACGCG-3'. Successfully-connected vectors were subjected to sequencing analysis(Shanghai Yingjun Co., China)[11]. Amplification of bacteria from the appropriate clones was accomplished by incubation followed by extraction of plasmid DNA. Table 1

Design of siRNA targeting rat CD40 gene

Target number

Sequence

1 2

5'-GCTCTTGAGAAGACCCAAT-3' 5'-AGGTGGTCAAGAAACCAAA-3'

3

5'- GGAACAAGTCAGACTGATA -3'

4

5'-TGGTAAAGAGAGTCGCATC-3'

Negative control(NC)

5'-TTCTCCGAACGTGTCACGT-3'

2.2 Construction of Rat CD40 Gene Eukaryotic Expression Vector According to the commercial cloning of rat CD40 gene(Wuhan Mitaka, China) , primer sequence that contains restriction endonuclease digestion sites and protective base was designed and synthesized by the company(Shanghai Biotechnology Co., China). DNA fragments of target gene were amplified through the PCR reaction, up(+): underlined position is EcoR I restriction site; down(–): underlined position is BamH1 restriction site. PCR product and the eukaryotic expression vector pEGFP-N1 were double, restrictively, digested with EcoR I and BamHI. T4-ligase reaction system was used for preparation of recombinant clone connecting liquid and transformation of

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competence E. coli and screen of positive clones. Positive clones that were successfully connected were subjected to sequencing analysis(Shanghai Yingjun Co., China). 2.3

Screening of Effective siRNA Target

Transient transfection into 293T cells, an established human renal epithelial cell line, is a convenient way to overexpress both cellular and extracellular proteins. The constructed rat CD40 eukaryotic expression vector and shRNA lentiviral frame plasmid with different targets were co-transfected into 293T cells with lipofectamine 2000(livitrogen) under good growing conditions. Effect of siRNAs on CD40 expression was assessed via the determination of the expression of CD40 protein by Western blot 48 h after transfection. The most effective target was selected for packaging lentiviral particles. The lentiviruses were used for the transfection of rat DCs and verification of RNAi efficacy. 2.4

Lentiviral Packaging and Titer Testing

293T cells in logarithmic phase were seeded into a 25 mL dish. Once cell fusion reached 70%, recombinant lentiviral frame plasmid and packaging helper plasmid were co-transfected into 293T cells(virus packaging cells) with the aid of calcium phosphate. Medium was replaced by fresh culture medium 12 h after transfection. The supernatant of 293T cell culture was collected and centrifuged(4000g) at 4 °C for 10 min to remove cell debris after 72 h transfection. Thereafter, the supernatant was filtered with a 0.45 µm filter(MillipQ) prior to super-centrifugation at 25000 r/min(Beckman V70ti) for 90 min. Virus precipitation was accomplished with ice PBS re-suspension solution. Finally, the virus particles were stored by split charging at 80 °C. A virus solution of 10 µL was subjected to 10-folds limited dilution and used to infect 293T cells. The viral titer was calculated by the determination of GFP fluorescence on a lentiviral vector 48 h after transfection. 2.5 Generation, Purification and Stimulation of Bone Marrow(BM)-derived DC Bone marrow(BM)-derived DC were propagated with some modifications as described somewhere[12]. Briefly, BM cells harvested from the femurs of normal Dark Agoulti rats were cultured in 6-well plates.

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Erythrocytes were lysed with 0.83%(mass fraction) NH4Cl buffer, and leukocytes depleted with antibodies on day 0. BM cells were subjected to gradient centrifugation with Ficoll liquid at 1500 r/min for 20 min to collect monocytes in the interfacial layer. Monocytes were centrifugally washed to obtain BM-derived monocytes. The cells were cultured in 24-well plates (2.5×106 cells/well) overnight at 37 °C in the atmosphere with 5% CO2 in RPMI-1640 complete medium (Life Technologies, Gaithersburg, MD) supplemented with antibodies, 10%(volume fraction) fetal calf serume(FBS). On day 1, non-adherent cells were removed carefully by gentle pipetting to obtain precursor DCs, to which fresh media containing 4.0 ng/mL rat recombinant GM-CSF and 4.0 ng/mL rat recombinant IL-4 were added. Cytokine-enriched medium was refreshed every 2 d, after gentle swirling of the plates, half of the old medium was aspirated and an equivalent volume of fresh, cytokine supplemented medium was added to the residue. Non-adherent cells released spontaneously from the clusters were harvested after 10 d. Cell viability was assessed by trypan blue staining. Some DCs were stimulated with 10 µg/mL LPS (Sigma) for 8 h. Assessment of phenotypical features of DCs was accomplished by flow cytometry and microscopy with the aid of antibodies against CD40, CD80, CD86 and MHC class II, respectively[10].

h, total RNA was extracted from cells with Trizol(Life Technologies, USA) according to the manufacturer’s instructions. A total of 2 μg of RNA was reverse transcribed in a 20 μL of reaction solution containing 10 units of M-MLV Reverse Transcriptase(Promega) and 0.5 μg of oligo(dT) primer. cDNA of 1 μL was used for real-time PCR. The MED19 gene was amplified with SYBR Master Mixture(Takara, Japan) on IQ5(BioRad, USA), comprised an initial denaturation at 95 °C for 15 s, then 45 cycles of denaturation at 95 °C for 5 s, 60 ºC for 30 s. The β-actin gene was used as an internal control for standardization in triplicate. PCR primer sequences were listed in Table 2. Table 2

Mature rat DCs with good growing condition were seeded into 6-well plates and cultured for 24 h. After addition of 10 µL of shRNA lentivirus(virus titer of 5×107 TU/mL) per well, mature rat DCs were incubated for another 72 h. GFP expression was observed under fluorescence microscopy to assess the efficiency of virus infection in rat DCs. Cells were stimulated by LPS for 8 h and subjected to RNA and protein sampling 96 h after transfection. Real-time PCR(BioRad, iQ5) and Western blot were used to assess the inhibitory effect of the lentiviral shRNA on CD40 gene expression at mRNA and protein levels in rat DCs. PCR primers were as follows: forward: 5'-CCAGTCGGCTTCTTCTCC-3'; reverse: 5'-GACCTCCAAGTTCTTATCTTCA-3'. 2.7

Real Time RT-PCR After transfection of DCs with lentiviruses for 96

PCR primer sequences

Primer Q-CD40-6F Q-CD40-6R

Sequence Forward: 5'-CCAGTCGGCTTCTTCTCC-3'; Reverse: 5'-GACCTCCAAGTTCTTATCTTCA-3'.

Actin-F

Forward: 5'-CCAGGCATTGCTGAGGG

Actin-R

Reverse:5'-GCTGGAAGGTGGACGTG-3'

The ΔCt value in quantitative analysis was calculated by subtracting the Ct(threshold cycle) value for β-actin from the Ct value for MED19 gene, and it was adopted as the expression level of CD40 gene. Furthermore, the ΔΔCt value was calculated by subtracting the ΔCt value in control groups and the expression change by RNAi was expressed as 2–ΔΔCt. 2.8

2.6 Quantification of Gene Silencing Efficacy in Rat DCs by Lentiviral-mediated CD40 shRNA

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Western Blot Analysis

After transfection for 96 h, the cells were lysed by pre-cooled lysis buffer and the protein content of the lysate was assessed. A 10% polyacrylamide gel with a 5% stack solution was poured. Then we loaded 30 μg for each well, and gel was run at 30 mA for 2 h. Transfer was semidry for 2 h in standard transfer buffer. The resulting membrane was blocked in 5% non-fat dry milk blocking buffer and then probed with protein-specific antibodies overnight at 4 °C. The membrane was then washed three times with TBST, followed by incubation for 2 h with appropriate secondary antibody at room temperature. After further washing, the membrane was developed by enhanced chemiluminescence(ECL, Amersham, UK). Goat anti-CD40(Abcam, UK) and mouse anti-GADHP (Santa Cruz, CA) were used. Secondary antibodies(anti-goat IgG and anti-mouse IgG) were used at a work concentration of 1:5000 for anti-goat IgG and 1:6000 for anti-mouse IgG(Santa Cruz, CA).

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Results

3.1 Construction of shRNA Lentiviral Expression Vector and CD40 Eukaryotic Expression Vector Connection of CD40 oligonucleotide sequence and lentiviral frame plasmids double-digested with the HpaI and XhoI was performed. The connecting product was transformed into E. coli. Positive recombinant clones were selected and identified with PCR. The sizes of PCR products of recombinant clones and unconnected shRNA empty vector were 384 bp and 322 bp, respectively. The successfully-connected clones were collected for sequence analysis. Sequencing data show that the insertion of the sequence of synthetic CD40 shRNA oligonucleotide chain was successfully performed[Fig.1(A)]. PCR fragment of

Fig.1

3.2

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CD40 gene and pEGF-PN1 vector were double digested. Directional connection of the digested products was performed after purification. The products were transformed into the competence cells. Their clones were identified with PCR. Their corresponding upstream and downstream primers were designed on the vector of the targeting gene. Size of the PCR product was 575 bp. Those PCR-positive clones identified indicate that CD40 shRNA oligonucleotide chain has been successfully inserted into pEGFP expression vector. Sequencing of successfully-connected clones shows that BLAST and CD40 mRNA coding sequence published in Genebank(NM_134360) are identical, indicating the success in the construction of eukaryotic expression vector for rat CD40 [Fig.1(B)].

Sequencing of lentiviral constructs

Identification of Effective Target of siRNA

Cotransfection of 293T cells and eukaryotic expression vector was performed, lentiviral siRNA vector with four targets derived from various regions of the CD40 coding sequence, and the negative control

lentivirus. Western blot shows that the No.2, and No.4 targets had 50% and more than 90% silencing efficiencies, respectively, while the negative control and the two remaining targets had no significant inhibition effect on CD40 expression(Fig.2). Based on these results, we selected No.4 target for packaging lentiviral shRNA plasmid, which showed the best activity of CD40 siRNA. 3.3 Packaging and Titer Determination of Lentiviral Vector

Fig.2

Western blot analysis of rat CD40 protein expression in 293T cells

Con: cells non-transfected; PC: cells co-transfected with CD40- overexpression vector and pcDNA3.1; NC: cells co-transfected with CD40- overexpression vector and negative-control-Lentivirus; 1#―4#: cells co-transfected with CD40-overexpression vector and CD40-RNAi(1#―4#)-Lentivirus.

Lentiviral vector system consists of three plasmids including shRNA expression component vector, helper vector with virus core protein gag/pol, and helper vector with VSVG virus membrane. The No. 4

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target shRNA vector and helper vector were applied to co-transfect 293T cells followed by microscopic observation of GFP expression. Virus titer was expressed

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as multiplied amounts of cells expressing GFP by corresponding dilution. The determined titer of 5×107 TU/mL indicates successful virus packaging(Fig.3).

Determination of virus titers

Microfluorographs of 293T cells after transfection represent titer of lentivirus. (A) 1 μL; (B) 10–1 μL; (C) 10–2 μL; (D) 10–3μL; (E) 10–4 μL; (F) 10–5 μL; (G) 10–6 μL; (H) 10–7 μL.

3.4 Transfection Efficiency of Rat DCS with the Lentivirus DCs were transfected with lentivirus according to multiplicity of infection(MOI) expressed as the number of lentivirus/number of cells. Assessment of infection rate was accomplished with the determination of the rate of positive expression of GFP by fluorescent microscopy by which a value was determined 10 as optimal MOI for 48 h after transfection. Cell transfection in 6-well plates was performed once cell fusion rate reached 70%. Addition of 10 µL of lentivirus per well, a titer of 5×107 TU/mL(Fig.4), can achieve a infection rate of more than 90% in DCs.

3.5

Silencing Efficacy of CD40 shRNA Lentivirus

Real time RT-PCR and Western blot were adopted to determine the effect of CD40 shRNA lentivirus on the expression of CD gene at mRNA and protein levels in DCs for 96 h after transfection with CON(mock transfection with PBS alone, i.e., non-transfected), NC(Negative control lentivirus) and RNAi(CD40 shRNA lentivirus), respectively. Real time-PCR shows that expression level of CD40

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Fig.4

Microfluorographs of DCs after transfection

DCs were infected with lentiviral CD40-siRNA(RNAi) and nonsilencing control lentivirus(NC), and saline(CON). (A1) and (A2) CON; (B1) and (B2) NC; (C1) and (C2) RNAi. (A1)―(C1) Fluorescent field; (A2)―(C2) bright field.

Lentivirus-mediated RNAi downregulates CD40 expression in DCs

(A) Total RNA was extracted and analyzed by real-time PCR. The level of CD40 mRNA expression was normalized with that of β-actin mRNA expression; (B) cell lysates were subjected to SDS-PAGE and immunoblotted with anti-CD40 and anti-GADPH antibody. GADPH was simultaneously immunodetected to verify the loading of similar amounts of cell lysates. ** p