Riments. doi:10.1371/journal.pone.0056110.gFigure 6. Efficient down regulation of HBsAg and HBeAg expression using linked shRNAs concatemers both in vitro and in vivo. (A) The diagram illustrates the principle of chaining two shRNAs derived from two different shRNA vectors into one vector. (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 (as a normalization control) per well. After 48 h, the HBsAg and HBeAg concentrations were determined. Means and standard deviations were generated from 3 independent experiments. (C) Serum HBsAg and (D) HBeAg were measured by quantitative ELISA at the indicated days after plasmids delivery. Groups of male C57/BL6 mice (n = 6) were intravenously injected with 6 mg pHBV1.18, 3 mg shRNA plasmids and 3 mg pSEAP2-Control (as an internal control). NCU is the abbreviation of “National Clinical Unit”. Statistical significance was determined respectively by comparing shRNAs groups with AS139-1819-3172. Due to limited serum resources, each sample was diluted 20-fold. doi:10.1371/journal.pone.0056110.gA Robust shRNA System Used for RNA Interferenceof biological systems, including treatment of various diseases. Finally, it would be highly beneficial to 307538-42-7 biological activity generate an economic and high-performance lentiviral vector system based on our shRNA method to carry out high-throughput genetic screens for loss-offunction phenotypes.collected and centrifuged at 12,0006g for 5 min to remove debris. Gluc activity was measured using BioLuxH Gaussia Luciferase Flex Assay Kit (New England Biolabs). To monitor transfection efficiency, cell supernatants were assayed for secreted alkaline phosphatase (SEAP) using a SEAP Reporter Assay Kit (Toyobo).Materials and Methods Ethics statementThe Medical Ethics Committee of Beijing Institute of Radiation Medicine specifically approved this study. All the male C57/BL6 mice used in this experiment received humane care. The hydrodynamic injection was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.3′-UTR Luciferase assayThe sequences containing the shRNAs target sites were cloned into a modified pGL3-control plasmid (pGL3M) as described previously[21,22]. HEK293T cells were co-transfected with 200 ng of pGL3M-UTR constructs and 200 ng shRNAs per well in 24-well plates GenJetTM Reagent (Ver. II). 100 ng pRL-CMV (Promega, Madison, WI) was co-transfected as the normalization control. Luciferase activity assays were performed 48 h post transfection using the dual luciferase reporter assay system (Promega).Plasmid constructionThe HBV replication-competent plasmids pHBV1.31 and pHBV1.18 containing 1.31 and 1.18 HBV genome copies, respectively, were constructed and preserved in our laboratory (unpublished data), pmWasabi-N was a gift from Allele Biotechnology Company, pSuper was obtained from Addgene (Cambridge, MA), vector pSEAP2-Control was a gift from Dr. Shuiping Liu (Central South University of China), and the pCMVGLuc vector was purchased from New England Biolabs (NEB, Ipswich, MA). To generate the pshOK-basic vector, the CMVmWasabi sequence present in pmWasabi-N was replaced with a CMV-emGFP cassette as a means of introducing respective restriction sites. In addition, a modified human Octapressin RNA-polymerase III H1 promoter was amplified from pSuper and cloned into the pCMV-emGFP generated above. Then, 2 short oligos containing 7 polyTs were annealed in NEB buffer 2 and.Riments. doi:10.1371/journal.pone.0056110.gFigure 6. Efficient down regulation of HBsAg and HBeAg expression using linked shRNAs concatemers both in vitro and in vivo. (A) The diagram illustrates the principle of chaining two shRNAs derived from two different shRNA vectors into one vector. (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 (as a normalization control) per well. After 48 h, the HBsAg and HBeAg concentrations were determined. Means and standard deviations were generated from 3 independent experiments. (C) Serum HBsAg and (D) HBeAg were measured by quantitative ELISA at the indicated days after plasmids delivery. Groups of male C57/BL6 mice (n = 6) were intravenously injected with 6 mg pHBV1.18, 3 mg shRNA plasmids and 3 mg pSEAP2-Control (as an internal control). NCU is the abbreviation of “National Clinical Unit”. Statistical significance was determined respectively by comparing shRNAs groups with AS139-1819-3172. Due to limited serum resources, each sample was diluted 20-fold. doi:10.1371/journal.pone.0056110.gA Robust shRNA System Used for RNA Interferenceof biological systems, including treatment of various diseases. Finally, it would be highly beneficial to generate an economic and high-performance lentiviral vector system based on our shRNA method to carry out high-throughput genetic screens for loss-offunction phenotypes.collected and centrifuged at 12,0006g for 5 min to remove debris. Gluc activity was measured using BioLuxH Gaussia Luciferase Flex Assay Kit (New England Biolabs). To monitor transfection efficiency, cell supernatants were assayed for secreted alkaline phosphatase (SEAP) using a SEAP Reporter Assay Kit (Toyobo).Materials and Methods Ethics statementThe Medical Ethics Committee of Beijing Institute of Radiation Medicine specifically approved this study. All the male C57/BL6 mice used in this experiment received humane care. The hydrodynamic injection was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.3′-UTR Luciferase assayThe sequences containing the shRNAs target sites were cloned into a modified pGL3-control plasmid (pGL3M) as described previously[21,22]. HEK293T cells were co-transfected with 200 ng of pGL3M-UTR constructs and 200 ng shRNAs per well in 24-well plates GenJetTM Reagent (Ver. II). 100 ng pRL-CMV (Promega, Madison, WI) was co-transfected as the normalization control. Luciferase activity assays were performed 48 h post transfection using the dual luciferase reporter assay system (Promega).Plasmid constructionThe HBV replication-competent plasmids pHBV1.31 and pHBV1.18 containing 1.31 and 1.18 HBV genome copies, respectively, were constructed and preserved in our laboratory (unpublished data), pmWasabi-N was a gift from Allele Biotechnology Company, pSuper was obtained from Addgene (Cambridge, MA), vector pSEAP2-Control was a gift from Dr. Shuiping Liu (Central South University of China), and the pCMVGLuc vector was purchased from New England Biolabs (NEB, Ipswich, MA). To generate the pshOK-basic vector, the CMVmWasabi sequence present in pmWasabi-N was replaced with a CMV-emGFP cassette as a means of introducing respective restriction sites. In addition, a modified human RNA-polymerase III H1 promoter was amplified from pSuper and cloned into the pCMV-emGFP generated above. Then, 2 short oligos containing 7 polyTs were annealed in NEB buffer 2 and.
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