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    New England Biolabs t5 exonuclease
    Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with <t>T5</t> exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t7 exonucleases
    Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with <t>T5</t> exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p
    T7 Exonucleases, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 4 article reviews
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    t7 exonucleases - by Bioz Stars, 2020-09
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    99
    New England Biolabs exonuclease t5
    In vitro HBV infection of rhesus macaque PH.  a  Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP.  b  Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488.  c  Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments.  d  HBV DNA qPCR on the same supernatants shown in  c . Each condition represents a single-biological sample ( N  = 1).  e  Total intracellular DNA from 1 × 10 6  rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates.  f  Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments.  g  Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4  or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)
    Exonuclease T5, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs phusion high fidelity dna polymerase
    In vitro HBV infection of rhesus macaque PH.  a  Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP.  b  Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488.  c  Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments.  d  HBV DNA qPCR on the same supernatants shown in  c . Each condition represents a single-biological sample ( N  = 1).  e  Total intracellular DNA from 1 × 10 6  rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates.  f  Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments.  g  Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4  or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 23530 article reviews
    Price from $9.99 to $1999.99
    phusion high fidelity dna polymerase - by Bioz Stars, 2020-09
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    Image Search Results


    Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with T5 exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing

    doi: 10.1016/j.omtn.2020.03.005

    Figure Lengend Snippet: Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with T5 exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p

    Article Snippet: To remove linear and RC-form HBV DNAs for Sanger and MiSeq sequencing of cccDNA, genomic DNAs were extracted and digested with T5 exonuclease (New England Biolabs) in the reaction mixture of 50 μL containing 500 ng of DNA, 5 μL of ,10× reaction buffer and 1 μL of T5 Exo at 37°C for 1 h, and afterward 11 mM EDTA was added to stop reaction.

    Techniques: Southern Blot, Infection, Countercurrent Chromatography, Sequencing, HBsAg Assay, Next-Generation Sequencing

    Construction outline of the MCP tac s promoter clusters . Fragment 5CP tac s with the flanking sequence was amplified by PCR with p5TG as the template. Fragment 1 was generated by digesting fragment 5CP tac s with  BamH I. Fragment 2 was digested from fragment 5CP tac s with  BamH I and  Hind III. Fragment 3 was linearized from the plasmid p5TG with  Hind III. Then, the three fragments were assembled together under the action of T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase in the isothermal process.

    Journal: Microbial Cell Factories

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

    doi: 10.1186/1475-2859-11-19

    Figure Lengend Snippet: Construction outline of the MCP tac s promoter clusters . Fragment 5CP tac s with the flanking sequence was amplified by PCR with p5TG as the template. Fragment 1 was generated by digesting fragment 5CP tac s with BamH I. Fragment 2 was digested from fragment 5CP tac s with BamH I and Hind III. Fragment 3 was linearized from the plasmid p5TG with Hind III. Then, the three fragments were assembled together under the action of T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase in the isothermal process.

    Article Snippet: Then, fragment 1, 2 and 3 were assembled together in vitro under the action of T5 exonuclease (Epicentre), Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Generated, Plasmid Preparation

    Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.

    Journal: Journal of Virology

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    doi: 10.1128/JVI.01117-18

    Figure Lengend Snippet: Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.

    Article Snippet: T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Techniques: Activity Assay, Cell Culture, Derivative Assay, Incubation, Southern Blot, Plasmid Preparation, Marker

    Titration analysis of T5 Exo and PSD. (A) Two micrograms of genomic DNA samples from HBV-free HepG2 hNTCP cells was incubated with T5 Exo or PSD in time-dependent (1, 2, 4, and 16 h) and dose-dependent (1 and 5 U) manners. After digestion, products were visualized on an agarose gel, and the expression level of the human β-globin gene was measured as a representative readout to show digestion degree of genomic DNA. (B) Two micrograms of pSHH2.1 plasmid was incubated with T5 Exo or PSD similarly, and the remaining plasmid in products was determined by pp466-541 or directly visualized on an agarose gel.

    Journal: Journal of Virology

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    doi: 10.1128/JVI.01117-18

    Figure Lengend Snippet: Titration analysis of T5 Exo and PSD. (A) Two micrograms of genomic DNA samples from HBV-free HepG2 hNTCP cells was incubated with T5 Exo or PSD in time-dependent (1, 2, 4, and 16 h) and dose-dependent (1 and 5 U) manners. After digestion, products were visualized on an agarose gel, and the expression level of the human β-globin gene was measured as a representative readout to show digestion degree of genomic DNA. (B) Two micrograms of pSHH2.1 plasmid was incubated with T5 Exo or PSD similarly, and the remaining plasmid in products was determined by pp466-541 or directly visualized on an agarose gel.

    Article Snippet: T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Techniques: Titration, Incubation, Agarose Gel Electrophoresis, Expressing, Plasmid Preparation

    T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Journal: Journal of Virology

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    doi: 10.1128/JVI.01117-18

    Figure Lengend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Article Snippet: T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Techniques: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    Detection of cccDNA and validation of Myrcludex B in 96-well plate format. (A) HepG2 hNTCP cells seeded in a 96-well plate were infected at an mge/cell of 1,000. Myrcludex B was coadministered, tenofovir was added postinoculation, and IFN-α-2a was applied during and after infection. (B) Cells were treated with each antiviral at eight doses (1:3.2 serial dilutions) in triplicates. (C) On day 7 p.i., DNA samples were extracted together using a vacuum-based system. Crude DNA in 100 μl of elute was ethanol precipitated and resuspended with 10 μl of water. T5 Exo digestion was performed prior to cccDNA quantification by PCR. HBeAg levels during days 5 to 7 p.i. in the supernatant in all wells were measured.

    Journal: Journal of Virology

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    doi: 10.1128/JVI.01117-18

    Figure Lengend Snippet: Detection of cccDNA and validation of Myrcludex B in 96-well plate format. (A) HepG2 hNTCP cells seeded in a 96-well plate were infected at an mge/cell of 1,000. Myrcludex B was coadministered, tenofovir was added postinoculation, and IFN-α-2a was applied during and after infection. (B) Cells were treated with each antiviral at eight doses (1:3.2 serial dilutions) in triplicates. (C) On day 7 p.i., DNA samples were extracted together using a vacuum-based system. Crude DNA in 100 μl of elute was ethanol precipitated and resuspended with 10 μl of water. T5 Exo digestion was performed prior to cccDNA quantification by PCR. HBeAg levels during days 5 to 7 p.i. in the supernatant in all wells were measured.

    Article Snippet: T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Techniques: Infection, Polymerase Chain Reaction

    cccDNA profiles in infections with increasing mge. (A) HepG2 hNTCP cells were infected with different amounts of virus inoculum (mge/cell of 30, 100, 300, 1,000, and 3,000) in parallel, and total DNA samples were prepared on day 10 p.i. Samples were hydrolyzed by T5 Exo (5 U, 60 min) at 37°C for 1 h, and cccDNA was determined using pp1040-1996. Total DNA copy numbers were also determined in undigested samples using pp466-541. (B) Within the same infections, secreted HBsAg values from day 7 to 10 p.i. were detected. (C) On day 10 p.i., intracellular HBcAg expression levels (red) were visualized. As a control to verify NTCP-mediated entry of the virus, Myrcludex B (1 μM) was administered during the infection.

    Journal: Journal of Virology

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    doi: 10.1128/JVI.01117-18

    Figure Lengend Snippet: cccDNA profiles in infections with increasing mge. (A) HepG2 hNTCP cells were infected with different amounts of virus inoculum (mge/cell of 30, 100, 300, 1,000, and 3,000) in parallel, and total DNA samples were prepared on day 10 p.i. Samples were hydrolyzed by T5 Exo (5 U, 60 min) at 37°C for 1 h, and cccDNA was determined using pp1040-1996. Total DNA copy numbers were also determined in undigested samples using pp466-541. (B) Within the same infections, secreted HBsAg values from day 7 to 10 p.i. were detected. (C) On day 10 p.i., intracellular HBcAg expression levels (red) were visualized. As a control to verify NTCP-mediated entry of the virus, Myrcludex B (1 μM) was administered during the infection.

    Article Snippet: T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Techniques: Infection, Expressing

    T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).

    Journal: Journal of Virology

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    doi: 10.1128/JVI.01117-18

    Figure Lengend Snippet: T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).

    Article Snippet: T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Techniques: Infection, Blocking Assay, Southern Blot, Polymerase Chain Reaction

    In vitro HBV infection of rhesus macaque PH.  a  Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP.  b  Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488.  c  Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments.  d  HBV DNA qPCR on the same supernatants shown in  c . Each condition represents a single-biological sample ( N  = 1).  e  Total intracellular DNA from 1 × 10 6  rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates.  f  Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments.  g  Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4  or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)

    Journal: Nature Communications

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

    doi: 10.1038/s41467-017-01953-y

    Figure Lengend Snippet: In vitro HBV infection of rhesus macaque PH. a Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP. b Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488. c Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments. d HBV DNA qPCR on the same supernatants shown in c . Each condition represents a single-biological sample ( N  = 1). e Total intracellular DNA from 1 × 10 6 rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates. f Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments. g Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4 or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)

    Article Snippet: DNA was eluted from NucleoSpin Tissue Columns with 100 μl pre-warmed (70 °C) buffer and subjected to T5 exonuclease treatment (New England Biolabs) at 37 °C for 30 min.

    Techniques: In Vitro, Infection, Labeling, Transduction, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Southern Blot, Purification, Marker