Inserted into the above-described vector containing a modified human H1 promoter. Two strategies were used to generate shRNA. The first used a single long oligonucleotide synthesized and annealed to form double strands of its self. The second approach used two short oligonucleotides and the 5′-end of the oligo containing the loop sequence was phosphorylated by T4 polynucleotide kinase in the presence of ATP. Then these two short oligonucleotides were annealed to form double strands. Plasmid pshOK-basic was digested using Sap I, gel-purified and ligated with the annealed products at 16 uC for 3 h using DNA Ligation Kit Ver.2.0 (Takara).HBsAg and HBeAg ELISAHepG2 cells were cotransfected with 200 ng of pHBV1.31, 200 ng shRNA plasmid and 100 ng pSEAP2-Control as a normalization control per well of 24-well tissue culture plates. Media was changed and collected 48 h after HBV plasmid transfection and supernatants centrifuged at 12,0006g for 5 min to remove debris before collection. The concentrations of HBsAg and HBeAg in the supernatants were determined by chemiluminescence using commercial assay kits (Tigsun Diagnostics Co., Ltd., Beijing, China).Transfection with shRNA vectors does not induce an interferon responseHepG2 cells were transfected with the shRNA vectors and untransfected cells were treated with 1000 IU of IFNa-2a (Shenyang Sunshine Pharmaceutical Company) for 24 h or left untreated. Total cellular RNA was prepared using TRIZOL (Invitrogen, Carlsbad, CA). Semi-quantitative RT-PCR using a 2step method was used to determine the mRNA expression level of several interferon inducible genes. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. Primers for a-actin, and the IFN-inducible genes MxA, 2′, 5′-oligoadenylate synthetase 1 (OAS1), signal transducer, activator of transcription 1 (STAT1), and interferon-stimulated gene 15 (ISG15) are described in reference [12].Cell culture and transfectionThe HepG2 and HEK293T cell lines were purchased from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China) and cultured in DMEM (Dulbecco’s Minimal Essential Media) medium containing 10 FBS (fetal bovine serum), 100 U/ml penicillin and 100 mg/ml streptomycin and maintained at 37 uC in a humidified 5 CO2 atmosphere. Plasmid cotransfections were carried out 16 h after seeding the cells using GenJetTM Reagent (Ver. II) (SignaGen Laboratories, Rockville, MD) following the manufacturer’s protocol.Animal and hydrodynamic transfectionMale C57/BL6 mice weighing 16?8 g (4? weeks old at the start of the experiments) were purchase LY-2409021 obtained and housed in the animal center of the Academy of Military Medicine Science. To evaluate the anti-viral effects of shRNAs in vivo, an HBV hydrodynamic injection was conducted. Briefly, purified HBV plasmid pHBV1.18 (6 mg), shRNA plasmids (3 mg) and the pSEAP2Control (3 ug) as an internal control were diluted in physiological saline in a volume equivalent to 10 of the body weight and then injected into the tail vein within 5? s. Sera was then assayed for HBsAg and HBeAg. For each group, six mice were used. All animals received humane care and the study protocol complied with the MC-LR institution’s ethics guidelines.Reporter gene assaysFor the b-gal assays, HepG2 cells were plated at 1.56105 cells per well in 24-well tissue culture plates (Nunc, Roskilde, Denmark) and cotransfected with 200 ng 1407003 of pAAV-LacZ (Stratagene), 200 ng shRNA plasmid and 100 ng pSEAP2-Control as a.Inserted into the above-described vector containing a modified human H1 promoter. Two strategies were used to generate shRNA. The first used a single long oligonucleotide synthesized and annealed to form double strands of its self. The second approach used two short oligonucleotides and the 5′-end of the oligo containing the loop sequence was phosphorylated by T4 polynucleotide kinase in the presence of ATP. Then these two short oligonucleotides were annealed to form double strands. Plasmid pshOK-basic was digested using Sap I, gel-purified and ligated with the annealed products at 16 uC for 3 h using DNA Ligation Kit Ver.2.0 (Takara).HBsAg and HBeAg ELISAHepG2 cells were cotransfected with 200 ng of pHBV1.31, 200 ng shRNA plasmid and 100 ng pSEAP2-Control as a normalization control per well of 24-well tissue culture plates. Media was changed and collected 48 h after HBV plasmid transfection and supernatants centrifuged at 12,0006g for 5 min to remove debris before collection. The concentrations of HBsAg and HBeAg in the supernatants were determined by chemiluminescence using commercial assay kits (Tigsun Diagnostics Co., Ltd., Beijing, China).Transfection with shRNA vectors does not induce an interferon responseHepG2 cells were transfected with the shRNA vectors and untransfected cells were treated with 1000 IU of IFNa-2a (Shenyang Sunshine Pharmaceutical Company) for 24 h or left untreated. Total cellular RNA was prepared using TRIZOL (Invitrogen, Carlsbad, CA). Semi-quantitative RT-PCR using a 2step method was used to determine the mRNA expression level of several interferon inducible genes. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. Primers for a-actin, and the IFN-inducible genes MxA, 2′, 5′-oligoadenylate synthetase 1 (OAS1), signal transducer, activator of transcription 1 (STAT1), and interferon-stimulated gene 15 (ISG15) are described in reference [12].Cell culture and transfectionThe HepG2 and HEK293T cell lines were purchased from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China) and cultured in DMEM (Dulbecco’s Minimal Essential Media) medium containing 10 FBS (fetal bovine serum), 100 U/ml penicillin and 100 mg/ml streptomycin and maintained at 37 uC in a humidified 5 CO2 atmosphere. Plasmid cotransfections were carried out 16 h after seeding the cells using GenJetTM Reagent (Ver. II) (SignaGen Laboratories, Rockville, MD) following the manufacturer’s protocol.Animal and hydrodynamic transfectionMale C57/BL6 mice weighing 16?8 g (4? weeks old at the start of the experiments) were obtained and housed in the animal center of the Academy of Military Medicine Science. To evaluate the anti-viral effects of shRNAs in vivo, an HBV hydrodynamic injection was conducted. Briefly, purified HBV plasmid pHBV1.18 (6 mg), shRNA plasmids (3 mg) and the pSEAP2Control (3 ug) as an internal control were diluted in physiological saline in a volume equivalent to 10 of the body weight and then injected into the tail vein within 5? s. Sera was then assayed for HBsAg and HBeAg. For each group, six mice were used. All animals received humane care and the study protocol complied with the institution’s ethics guidelines.Reporter gene assaysFor the b-gal assays, HepG2 cells were plated at 1.56105 cells per well in 24-well tissue culture plates (Nunc, Roskilde, Denmark) and cotransfected with 200 ng 1407003 of pAAV-LacZ (Stratagene), 200 ng shRNA plasmid and 100 ng pSEAP2-Control as a.
