Suppress HBsAg and HBeAg expression in the absence of detectable shRNA-related interferon responses (Fig. S5). However, we didn’t observe differences in knockdown efficiency enhancement following the combination of 2 or 3 shRNAs in vitro, suggesting that this phenomenon might be caused by promoter disturbances or competition. An indirect experiment using the 3′-UTR luciferase assay was used to demonstrate that the silencing activity of AS139-1819-3172 were indeed some lower comparing with AS139, AS1819 and 3172 respcetively (Fig. S6). We also found that the co-transfected multiple shRNA constructs had better silencing activity than the shRNA plasmid with multiple shRNAs when the same amount of each shRNA scaffold was used (Fig. S7). This verified our speculation that multiple H1 promoter in the same vector may interference with each other. Regardless, the three connected shRNA structure was shown to efficiently inhibit HBV antigens expression especially in vivo.shRNA system demonstrated here gave us an acceptable and more economical way to knockdown genes. At the last, two different shRNA clone methods were compared and the two short oligonucleotides based shRNA construction GSK2879552 web method was more efficient than the single long oligonucleotide based strategy. The single long oligo would be prone to form hairpin structure itself, and this could affect the double strands formation. We think this is the reason why the two short oligos method was superior to the single long oligo strategy. With shorter oligos, the error rate of synthesis was also decreased.ConclusionsWe describe a simple and robust shRNA construction system that will enable users to easily construct single or multiple shRNAs efficiently at a low cost. Using this method, we systemically screened the target sites for HBV knockdown and successfully depressed HBV antigen expression with connected multiple shRNAs both in vitro and in vivo. The method described here provides an inexpensive and powerful new tool with the potential of down regulating gene expression that can be applied to a varietyFigure 4. Suppression of two reporter genes by the shRNAs cloned with our methods. (A) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pAAV-LacZ, 200 ng shRNA plasmid and 100 ng pSEAP2-Control (used as a normalization control). LacZ was stained and photographed 48 h after cotransfection of HepG2 cells. Magnification 6200. The scale 18325633 bar represents 1 mm. (B) The same procedure described above was carried out using pCMV-Gluc in place of pAAV-LacZ. After 48 h, Gluc activity was determined. An shRNA scaffold (targeted to GUCUCCACGCGCAGUACAUUU) irrelevant to any known human or mouse gene sequence was designed as the negative control (“neg”) [23,24]. Means and standard deviations were generated from 3 independent experiments. doi:10.1371/journal.pone.0056110.gA Robust shRNA System Used for RNA InterferenceFigure 5. Screening of shRNAs for significant suppression of HBsAg and HBeAg. (A) shRNAs targeting to the conserved regions of HBV genome were designed and illustrated. The numbers represent nucleotide (nt) coordinates MedChemExpress GSK2126458 relative to the HBV (genotype B) pgRNA start site. (B) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pHBV1.31, 200 ng shRNA plasmid and 100 ng pSEAP2-Control per well. The HBsAg and HBeAg concentrations in cell supernatants were detected 48 h post transfection. Means and standard deviations were generated from 3 independent expe.Suppress HBsAg and HBeAg expression in the absence of detectable shRNA-related interferon responses (Fig. S5). However, we didn’t observe differences in knockdown efficiency enhancement following the combination of 2 or 3 shRNAs in vitro, suggesting that this phenomenon might be caused by promoter disturbances or competition. An indirect experiment using the 3′-UTR luciferase assay was used to demonstrate that the silencing activity of AS139-1819-3172 were indeed some lower comparing with AS139, AS1819 and 3172 respcetively (Fig. S6). We also found that the co-transfected multiple shRNA constructs had better silencing activity than the shRNA plasmid with multiple shRNAs when the same amount of each shRNA scaffold was used (Fig. S7). This verified our speculation that multiple H1 promoter in the same vector may interference with each other. Regardless, the three connected shRNA structure was shown to efficiently inhibit HBV antigens expression especially in vivo.shRNA system demonstrated here gave us an acceptable and more economical way to knockdown genes. At the last, two different shRNA clone methods were compared and the two short oligonucleotides based shRNA construction method was more efficient than the single long oligonucleotide based strategy. The single long oligo would be prone to form hairpin structure itself, and this could affect the double strands formation. We think this is the reason why the two short oligos method was superior to the single long oligo strategy. With shorter oligos, the error rate of synthesis was also decreased.ConclusionsWe describe a simple and robust shRNA construction system that will enable users to easily construct single or multiple shRNAs efficiently at a low cost. Using this method, we systemically screened the target sites for HBV knockdown and successfully depressed HBV antigen expression with connected multiple shRNAs both in vitro and in vivo. The method described here provides an inexpensive and powerful new tool with the potential of down regulating gene expression that can be applied to a varietyFigure 4. Suppression of two reporter genes by the shRNAs cloned with our methods. (A) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pAAV-LacZ, 200 ng shRNA plasmid and 100 ng pSEAP2-Control (used as a normalization control). LacZ was stained and photographed 48 h after cotransfection of HepG2 cells. Magnification 6200. The scale 18325633 bar represents 1 mm. (B) The same procedure described above was carried out using pCMV-Gluc in place of pAAV-LacZ. After 48 h, Gluc activity was determined. An shRNA scaffold (targeted to GUCUCCACGCGCAGUACAUUU) irrelevant to any known human or mouse gene sequence was designed as the negative control (“neg”) [23,24]. Means and standard deviations were generated from 3 independent experiments. doi:10.1371/journal.pone.0056110.gA Robust shRNA System Used for RNA InterferenceFigure 5. Screening of shRNAs for significant suppression of HBsAg and HBeAg. (A) shRNAs targeting to the conserved regions of HBV genome were designed and illustrated. The numbers represent nucleotide (nt) coordinates relative to the HBV (genotype B) pgRNA start site. (B) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pHBV1.31, 200 ng shRNA plasmid and 100 ng pSEAP2-Control per well. The HBsAg and HBeAg concentrations in cell supernatants were detected 48 h post transfection. Means and standard deviations were generated from 3 independent expe.