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    TaKaRa plvx ires zsgreen1
    ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with <t>pLVX-ORF2-IRES-zsGreen</t> and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P
    Plvx Ires Zsgreen1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa plvx ef1α ires zsgreen1
    ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with <t>pLVX-ORF2-IRES-zsGreen</t> and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P
    Plvx Ef1α Ires Zsgreen1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles

    doi: 10.1073/pnas.1614955114

    Figure Lengend Snippet: ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P

    Article Snippet: To construct a lentiviral construct that encodes Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3, the Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3 cDNA was amplified by PCR from the HEV Kernow C1/p6 construct (a kind gift from Suzanne Emerson, NIH, Bethesda, MD) and then cloned into pLVX-IRES-zsGreen1 or pLEX-IRES-mCherry vectors using the In-Fusion HD Cloning Kit (Clontech).

    Techniques: Flow Cytometry, Cytometry, Expressing, Transduction, Infection, Cell Culture, Incubation, Luciferase, Activity Assay, Transfection

    HEV ORF1 is able to function in trans to replicate HEV RNA. (a) Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1 expression. HepG2C3A cells were transduced with pLVX-ORF1-IRES-zsGreen (wild type [wt] or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. FSC, forward scatter. (c) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells, or HepG2C3A cells transduced with HEV ORF1 or its GAD mutant, were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or RNA from its mutants, and Gaussia luciferase (Gluc) activity was quantified. Values are means plus standard deviations (SD) (error bars) ( n = 3). Values that are significantly different ( P

    Journal: mBio

    Article Title: Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

    doi: 10.1128/mBio.00769-18

    Figure Lengend Snippet: HEV ORF1 is able to function in trans to replicate HEV RNA. (a) Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1 expression. HepG2C3A cells were transduced with pLVX-ORF1-IRES-zsGreen (wild type [wt] or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. FSC, forward scatter. (c) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells, or HepG2C3A cells transduced with HEV ORF1 or its GAD mutant, were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or RNA from its mutants, and Gaussia luciferase (Gluc) activity was quantified. Values are means plus standard deviations (SD) (error bars) ( n = 3). Values that are significantly different ( P

    Article Snippet: To construct lentiviral constructs encoding ORF1 of Kernow C1/p6 (GenBank accession number JQ679013 ), the Kernow C1/p6 ORF1 cDNA was amplified by PCR from a plasmid encoding the full-length (FL) infectious HEV clone Kernow C1/p6 (a kind gift from Suzanne Emerson, NIH) and then cloned into pLVX-IRES-zsGreen1 vector using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Expressing, Transduction, Mutagenesis, Cell Culture, Luciferase, Activity Assay

    The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

    Journal: BMC Cancer

    Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

    doi: 10.1186/s12885-018-4097-z

    Figure Lengend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

    Article Snippet: To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively.

    Techniques: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry