Methods HBV Plasmids Five HBV 1.35-fold genome plasmids – N10 (genotype Ae, AY707087), C4371 (genotype Ba, GU357842), Y1021 (genotype C1, GU357845), Y10 (genotype D1, GU357846) and W29 (genotype I1, GU357844) were used for transfection and hydrodynamic injection. The constructions and molecular

and phenotypic characteristics are described in our previous report [36]. Bioinformatics Analysis To define the conservative sites on HBV genomes amongst the various genotypes, all available complete genome sequences #PSI-7977 cost randurls[1|1|,|CHEM1|]# of HBV, as of April 2009, were downloaded from GenBank. Multiple alignment was done with ClustalX2 under default settings (Gap Opening:10, Gap Extension: 0.2, Delay Divergent Sequences(%): 30, DNA Transition Weight: 0.5, Use Negative Matrix: Off). The most representative and informative sequence in terms of phylogeny were collected as a dataset and the most similar sequences were removed using all

pairwise distance scan. A total of 327 HBV genomes including A-I genotypes and nearly all reported subtypes were remained in the final dataset. The genotypes and subtypes of six HBV genomes isolated in the study were submitted to phylogenetic analysis using MEGA 4.0 software (data not shown). Forty sites with conservative sequences were selected and the shRNA plasmids were constructed (Table 1). The designed siRNA were evaluated for potential off-target effects by the online SOS program http://​rnai.​cs.​unm.​edu/​offTarget. The sequences and positions Belnacasan price of the forty designed shRNA targets are shown in Table 1. ShRNA Plasmids ShRNA plasmids were cloned downstream of the human H1 promoter in the vector pSUPER [37]. The target sites for siRNA were

chosen based either on conservative sites among the major HBV genotypes and subtypes. An shRNA plasmid targeting the firefly luciferase gene was used as a control (L1254: TGG CTA CAT TCT GGA GAC ATA). Cell Culture and In Vitro Transfection The plasmids used for in vitro transfection were purified with PlasmidSelect Xtra Starter Kit (GE Health, Sweden) and the concentrations were determined by the UV-spectrophotometric method. To determine the ability of siRNA to inhibit HBV gene expression in cell cultures, Huh7 cells were co-transfected with 4 μg of HBV plasmids, 1 μg of shRNA plasmids and 0.4 μg of a pcDNA3.1-SEAP plasmid using Lipofectamine 2000 (Invitrogen, Shanghai, China) following the manufacturer’s instructions. They were then harvested four days later. The pcDNA3.1-SEAP plasmid is a reporter plasmid expressing secreted alkaline phosphatase and used for transfection efficiency standardization by estimating SEAP enzymatic activity (Pierce; Kunming, China) in the culture supernatant.