psv40 cluc  (New England Biolabs)


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    Name:
    pSV40 CLuc Control Plasmid
    Description:
    pSV40 CLuc Control Plasmid 20 ug
    Catalog Number:
    n0318s
    Price:
    297
    Size:
    20 ug
    Category:
    Cellular Biology
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    Structured Review

    New England Biolabs psv40 cluc
    pSV40 CLuc Control Plasmid
    pSV40 CLuc Control Plasmid 20 ug
    https://www.bioz.com/result/psv40 cluc/product/New England Biolabs
    Average 94 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    psv40 cluc - by Bioz Stars, 2021-02
    94/100 stars

    Images

    1) Product Images from "In Vitro Screening for Compounds That Enhance Human L1 Mobilization"

    Article Title: In Vitro Screening for Compounds That Enhance Human L1 Mobilization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074629

    The dual secreted luciferase L1 retrotransposition assay. (A) Timeline of dual secreted luciferase L1 retrotransposition assay. HeLa cells transfected with 99-PUR-RPS- mGLucI and pSV40-CLuc were exposed to a test compound and puromycin for 2 days. The cells were then exposed to the test compound alone for an additional 3 days. GLuc and CLuc luminescence in the medium and cellular ATP were measured with a luminometer. GLuc luminescence indicates L1 retrotransposition activity. CLuc luminescence was used for normalization as an internal control. Cell viability was evaluated by cellular ATP content. (B) Effect of JM111 (L1 RP ORF1 mutant) on L1 retrotransposition activity. Either 99-PUR-RPS- mGLucI or 99-PUR -JM111- mGLucI containing two missense mutations in L1 RP ORF1 and pSV40-CLuc were co-transfected. L1 retrotransposition activity is represented as normalized luminescence. *** p
    Figure Legend Snippet: The dual secreted luciferase L1 retrotransposition assay. (A) Timeline of dual secreted luciferase L1 retrotransposition assay. HeLa cells transfected with 99-PUR-RPS- mGLucI and pSV40-CLuc were exposed to a test compound and puromycin for 2 days. The cells were then exposed to the test compound alone for an additional 3 days. GLuc and CLuc luminescence in the medium and cellular ATP were measured with a luminometer. GLuc luminescence indicates L1 retrotransposition activity. CLuc luminescence was used for normalization as an internal control. Cell viability was evaluated by cellular ATP content. (B) Effect of JM111 (L1 RP ORF1 mutant) on L1 retrotransposition activity. Either 99-PUR-RPS- mGLucI or 99-PUR -JM111- mGLucI containing two missense mutations in L1 RP ORF1 and pSV40-CLuc were co-transfected. L1 retrotransposition activity is represented as normalized luminescence. *** p

    Techniques Used: Luciferase, Transfection, Activity Assay, Mutagenesis

    2) Product Images from "Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation"

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19108

    Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p
    Figure Legend Snippet: Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p

    Techniques Used: Transfection, Luciferase, Derivative Assay, Activity Assay, Reporter Assay, Infection, Expressing, Quantitative RT-PCR, Concentration Assay, Cell Viability Assay

    Related Articles

    Clone Assay:

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Subgenomic replicon plasmids of the Con1 strain (HCV genotype 1b), pFK-I389Luci/NS3-3’/NK5.1 [ ], were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany). pSGR-I389/GLuc was constructed by substitution of GLuc into the Luc gene of pFK-I389Luci/NS3-3’/NK5.1. pSGR-I389/GLuc-GND was constructed by introduction of a mutation in NS5B by PCR using the primers: 5’-CCGTGTTGAGGAGTCAATCTAC-3’(forward) and 5’-GTGTCTAGCTGTCTCCCACG-3’ (reverse). pSGR-JFH1, pHH/SGR-JFH1/GLuc, pHH/SGR-JFH1/GLuc/GND, pcDNA3.1-CD81, pRLucHCVLuc (H77 IRES; HCV genotype 1a), and pRLucEMCVLuc (EMCV IRES) were constructed as previously described [ – ]. pSV40-CLuc was purchased from New England Biolabs. pNL4-3.Luc.R-.E- was obtained from Dr. Niveen Mulholland through the National Institutes of Health AIDS Reagent Program (Bethesda, MD, USA). pNL4-3.NanoLuc.R−.E− was constructed by substitution of NanoLuc (pNL1.1; Promega, Madison, WI, USA) into the Luc gene of pNL4-3.Luc.R-.E-. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was digested with Pme I and Xba I and the fragment was recombined with annealed DNA fragments for miRNA target site and its mutant by the In-Fusion HD cloning kit (Takara, Shiga, Japan). ..

    Transfection:

    Article Title: The proto-oncogene Myc drives expression of the NK cell-activating NKp30 ligand B7-H6 in tumor cells
    Article Snippet: .. Normalization was performed by dividing the relative light unit (RLU) values obtained after transfection with the pGLuc-basic vector with the RLU values obtained with the pSV40-Cluc vector. .. ChIP was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer's instructions.

    Luciferase:

    Article Title: JC Polyomavirus Uses Extracellular Vesicles To Infect Target Cells
    Article Snippet: .. Luciferase reporter vector pSV40_CLuc expresses a secreted form of Cypridina luciferase under the control of SV40 early promoter (New England Biolabs, Ipswich, MA). .. Mock pseudovirus was produced by the same method, substituting nonspecific DNA for the viral expression plasmids.

    Article Title: In Vitro Screening for Compounds That Enhance Human L1 Mobilization
    Article Snippet: .. The JM111 mutant contains two missense mutations in L1RP ORF1 that abolish retrotransposition . pCMV-GLuc (New England BioLabs) constitutively expresses Gaussia luciferase under a CMV promoter. pSV40-CLuc (New England BioLabs), which contains an SV40 promoter and Cypridina luciferase (CLuc) , was used as an internal control vector for the high-throughput 96-well retrotransposition assay. .. Ultimate ORF cDNA clones (Invitrogen) were V5-tagged on their N-termini by shuttling them from pENTR221 vector into pcDNA3.1/nV5-DEST using Gateway Technology (Invitrogen) as previously described .

    Reporter Assay:

    Article Title: Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist
    Article Snippet: .. Reporter assay the pGluc-NF-κB construct was obtained from Genecopoeia (Rockville, MD, USA). pSV40-CLuc control plasmid was obtained from NEB (Ipswich, MA, USA). .. These constructs were co-transfected to 293 cells by FugeneHD (Promega).

    Mutagenesis:

    Article Title: In Vitro Screening for Compounds That Enhance Human L1 Mobilization
    Article Snippet: .. The JM111 mutant contains two missense mutations in L1RP ORF1 that abolish retrotransposition . pCMV-GLuc (New England BioLabs) constitutively expresses Gaussia luciferase under a CMV promoter. pSV40-CLuc (New England BioLabs), which contains an SV40 promoter and Cypridina luciferase (CLuc) , was used as an internal control vector for the high-throughput 96-well retrotransposition assay. .. Ultimate ORF cDNA clones (Invitrogen) were V5-tagged on their N-termini by shuttling them from pENTR221 vector into pcDNA3.1/nV5-DEST using Gateway Technology (Invitrogen) as previously described .

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Subgenomic replicon plasmids of the Con1 strain (HCV genotype 1b), pFK-I389Luci/NS3-3’/NK5.1 [ ], were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany). pSGR-I389/GLuc was constructed by substitution of GLuc into the Luc gene of pFK-I389Luci/NS3-3’/NK5.1. pSGR-I389/GLuc-GND was constructed by introduction of a mutation in NS5B by PCR using the primers: 5’-CCGTGTTGAGGAGTCAATCTAC-3’(forward) and 5’-GTGTCTAGCTGTCTCCCACG-3’ (reverse). pSGR-JFH1, pHH/SGR-JFH1/GLuc, pHH/SGR-JFH1/GLuc/GND, pcDNA3.1-CD81, pRLucHCVLuc (H77 IRES; HCV genotype 1a), and pRLucEMCVLuc (EMCV IRES) were constructed as previously described [ – ]. pSV40-CLuc was purchased from New England Biolabs. pNL4-3.Luc.R-.E- was obtained from Dr. Niveen Mulholland through the National Institutes of Health AIDS Reagent Program (Bethesda, MD, USA). pNL4-3.NanoLuc.R−.E− was constructed by substitution of NanoLuc (pNL1.1; Promega, Madison, WI, USA) into the Luc gene of pNL4-3.Luc.R-.E-. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was digested with Pme I and Xba I and the fragment was recombined with annealed DNA fragments for miRNA target site and its mutant by the In-Fusion HD cloning kit (Takara, Shiga, Japan). ..

    Construct:

    Article Title: Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist
    Article Snippet: .. Reporter assay the pGluc-NF-κB construct was obtained from Genecopoeia (Rockville, MD, USA). pSV40-CLuc control plasmid was obtained from NEB (Ipswich, MA, USA). .. These constructs were co-transfected to 293 cells by FugeneHD (Promega).

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Subgenomic replicon plasmids of the Con1 strain (HCV genotype 1b), pFK-I389Luci/NS3-3’/NK5.1 [ ], were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany). pSGR-I389/GLuc was constructed by substitution of GLuc into the Luc gene of pFK-I389Luci/NS3-3’/NK5.1. pSGR-I389/GLuc-GND was constructed by introduction of a mutation in NS5B by PCR using the primers: 5’-CCGTGTTGAGGAGTCAATCTAC-3’(forward) and 5’-GTGTCTAGCTGTCTCCCACG-3’ (reverse). pSGR-JFH1, pHH/SGR-JFH1/GLuc, pHH/SGR-JFH1/GLuc/GND, pcDNA3.1-CD81, pRLucHCVLuc (H77 IRES; HCV genotype 1a), and pRLucEMCVLuc (EMCV IRES) were constructed as previously described [ – ]. pSV40-CLuc was purchased from New England Biolabs. pNL4-3.Luc.R-.E- was obtained from Dr. Niveen Mulholland through the National Institutes of Health AIDS Reagent Program (Bethesda, MD, USA). pNL4-3.NanoLuc.R−.E− was constructed by substitution of NanoLuc (pNL1.1; Promega, Madison, WI, USA) into the Luc gene of pNL4-3.Luc.R-.E-. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was digested with Pme I and Xba I and the fragment was recombined with annealed DNA fragments for miRNA target site and its mutant by the In-Fusion HD cloning kit (Takara, Shiga, Japan). ..

    High Throughput Screening Assay:

    Article Title: In Vitro Screening for Compounds That Enhance Human L1 Mobilization
    Article Snippet: .. The JM111 mutant contains two missense mutations in L1RP ORF1 that abolish retrotransposition . pCMV-GLuc (New England BioLabs) constitutively expresses Gaussia luciferase under a CMV promoter. pSV40-CLuc (New England BioLabs), which contains an SV40 promoter and Cypridina luciferase (CLuc) , was used as an internal control vector for the high-throughput 96-well retrotransposition assay. .. Ultimate ORF cDNA clones (Invitrogen) were V5-tagged on their N-termini by shuttling them from pENTR221 vector into pcDNA3.1/nV5-DEST using Gateway Technology (Invitrogen) as previously described .

    Generated:

    Article Title: Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus
    Article Snippet: .. Mock pseudovirus controls were generated by transfecting 293FT cells with non-specific DNA (ns-DNA) and the pSV40-Cluc (NEB # N0318) reporter plasmid in a 5:1 ratio and following the same production/purification protocol as pseudovirus. .. As the viral structural proteins are expressed, they assemble into pseudovirus particles and package available plasmid DNA.

    Article Title: Phosphoinositide 3′-Kinase γ Facilitates Polyomavirus Infection
    Article Snippet: .. Mock pseudovirus controls were generated by transfecting 293FT cells with non-specific DNA and pSV40-Cluc in a 5:1 ratio and following the same production/purification protocol as JCPsV. .. Similar procedures were used to produce BK polyomavirus and Merkel Cell polyomavirus pseudoviruses expressing Gaussia luciferase (BKPsV-Gluc and MCPsV-Gluc, respectively).

    Expressing:

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Subgenomic replicon plasmids of the Con1 strain (HCV genotype 1b), pFK-I389Luci/NS3-3’/NK5.1 [ ], were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany). pSGR-I389/GLuc was constructed by substitution of GLuc into the Luc gene of pFK-I389Luci/NS3-3’/NK5.1. pSGR-I389/GLuc-GND was constructed by introduction of a mutation in NS5B by PCR using the primers: 5’-CCGTGTTGAGGAGTCAATCTAC-3’(forward) and 5’-GTGTCTAGCTGTCTCCCACG-3’ (reverse). pSGR-JFH1, pHH/SGR-JFH1/GLuc, pHH/SGR-JFH1/GLuc/GND, pcDNA3.1-CD81, pRLucHCVLuc (H77 IRES; HCV genotype 1a), and pRLucEMCVLuc (EMCV IRES) were constructed as previously described [ – ]. pSV40-CLuc was purchased from New England Biolabs. pNL4-3.Luc.R-.E- was obtained from Dr. Niveen Mulholland through the National Institutes of Health AIDS Reagent Program (Bethesda, MD, USA). pNL4-3.NanoLuc.R−.E− was constructed by substitution of NanoLuc (pNL1.1; Promega, Madison, WI, USA) into the Luc gene of pNL4-3.Luc.R-.E-. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was digested with Pme I and Xba I and the fragment was recombined with annealed DNA fragments for miRNA target site and its mutant by the In-Fusion HD cloning kit (Takara, Shiga, Japan). ..

    Polymerase Chain Reaction:

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Subgenomic replicon plasmids of the Con1 strain (HCV genotype 1b), pFK-I389Luci/NS3-3’/NK5.1 [ ], were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany). pSGR-I389/GLuc was constructed by substitution of GLuc into the Luc gene of pFK-I389Luci/NS3-3’/NK5.1. pSGR-I389/GLuc-GND was constructed by introduction of a mutation in NS5B by PCR using the primers: 5’-CCGTGTTGAGGAGTCAATCTAC-3’(forward) and 5’-GTGTCTAGCTGTCTCCCACG-3’ (reverse). pSGR-JFH1, pHH/SGR-JFH1/GLuc, pHH/SGR-JFH1/GLuc/GND, pcDNA3.1-CD81, pRLucHCVLuc (H77 IRES; HCV genotype 1a), and pRLucEMCVLuc (EMCV IRES) were constructed as previously described [ – ]. pSV40-CLuc was purchased from New England Biolabs. pNL4-3.Luc.R-.E- was obtained from Dr. Niveen Mulholland through the National Institutes of Health AIDS Reagent Program (Bethesda, MD, USA). pNL4-3.NanoLuc.R−.E− was constructed by substitution of NanoLuc (pNL1.1; Promega, Madison, WI, USA) into the Luc gene of pNL4-3.Luc.R-.E-. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was digested with Pme I and Xba I and the fragment was recombined with annealed DNA fragments for miRNA target site and its mutant by the In-Fusion HD cloning kit (Takara, Shiga, Japan). ..

    Plasmid Preparation:

    Article Title: JC Polyomavirus Uses Extracellular Vesicles To Infect Target Cells
    Article Snippet: .. Luciferase reporter vector pSV40_CLuc expresses a secreted form of Cypridina luciferase under the control of SV40 early promoter (New England Biolabs, Ipswich, MA). .. Mock pseudovirus was produced by the same method, substituting nonspecific DNA for the viral expression plasmids.

    Article Title: Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus
    Article Snippet: .. Mock pseudovirus controls were generated by transfecting 293FT cells with non-specific DNA (ns-DNA) and the pSV40-Cluc (NEB # N0318) reporter plasmid in a 5:1 ratio and following the same production/purification protocol as pseudovirus. .. As the viral structural proteins are expressed, they assemble into pseudovirus particles and package available plasmid DNA.

    Article Title: In Vitro Screening for Compounds That Enhance Human L1 Mobilization
    Article Snippet: .. The JM111 mutant contains two missense mutations in L1RP ORF1 that abolish retrotransposition . pCMV-GLuc (New England BioLabs) constitutively expresses Gaussia luciferase under a CMV promoter. pSV40-CLuc (New England BioLabs), which contains an SV40 promoter and Cypridina luciferase (CLuc) , was used as an internal control vector for the high-throughput 96-well retrotransposition assay. .. Ultimate ORF cDNA clones (Invitrogen) were V5-tagged on their N-termini by shuttling them from pENTR221 vector into pcDNA3.1/nV5-DEST using Gateway Technology (Invitrogen) as previously described .

    Article Title: The proto-oncogene Myc drives expression of the NK cell-activating NKp30 ligand B7-H6 in tumor cells
    Article Snippet: .. Normalization was performed by dividing the relative light unit (RLU) values obtained after transfection with the pGLuc-basic vector with the RLU values obtained with the pSV40-Cluc vector. .. ChIP was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer's instructions.

    Article Title: Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist
    Article Snippet: .. Reporter assay the pGluc-NF-κB construct was obtained from Genecopoeia (Rockville, MD, USA). pSV40-CLuc control plasmid was obtained from NEB (Ipswich, MA, USA). .. These constructs were co-transfected to 293 cells by FugeneHD (Promega).

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Subgenomic replicon plasmids of the Con1 strain (HCV genotype 1b), pFK-I389Luci/NS3-3’/NK5.1 [ ], were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany). pSGR-I389/GLuc was constructed by substitution of GLuc into the Luc gene of pFK-I389Luci/NS3-3’/NK5.1. pSGR-I389/GLuc-GND was constructed by introduction of a mutation in NS5B by PCR using the primers: 5’-CCGTGTTGAGGAGTCAATCTAC-3’(forward) and 5’-GTGTCTAGCTGTCTCCCACG-3’ (reverse). pSGR-JFH1, pHH/SGR-JFH1/GLuc, pHH/SGR-JFH1/GLuc/GND, pcDNA3.1-CD81, pRLucHCVLuc (H77 IRES; HCV genotype 1a), and pRLucEMCVLuc (EMCV IRES) were constructed as previously described [ – ]. pSV40-CLuc was purchased from New England Biolabs. pNL4-3.Luc.R-.E- was obtained from Dr. Niveen Mulholland through the National Institutes of Health AIDS Reagent Program (Bethesda, MD, USA). pNL4-3.NanoLuc.R−.E− was constructed by substitution of NanoLuc (pNL1.1; Promega, Madison, WI, USA) into the Luc gene of pNL4-3.Luc.R-.E-. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was digested with Pme I and Xba I and the fragment was recombined with annealed DNA fragments for miRNA target site and its mutant by the In-Fusion HD cloning kit (Takara, Shiga, Japan). ..

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    New England Biolabs psv40 cluc
    The dual secreted luciferase L1 retrotransposition assay. (A) Timeline of dual secreted luciferase L1 retrotransposition assay. HeLa cells transfected with 99-PUR-RPS- mGLucI and <t>pSV40-CLuc</t> were exposed to a test compound and puromycin for 2 days. The cells were then exposed to the test compound alone for an additional 3 days. GLuc and CLuc luminescence in the medium and cellular ATP were measured with a luminometer. GLuc luminescence indicates L1 retrotransposition activity. CLuc luminescence was used for normalization as an internal control. Cell viability was evaluated by cellular ATP content. (B) Effect of JM111 (L1 RP ORF1 mutant) on L1 retrotransposition activity. Either 99-PUR-RPS- mGLucI or 99-PUR -JM111- mGLucI containing two missense mutations in L1 RP ORF1 and pSV40-CLuc were co-transfected. L1 retrotransposition activity is represented as normalized luminescence. *** p
    Psv40 Cluc, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/psv40 cluc/product/New England Biolabs
    Average 94 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    psv40 cluc - by Bioz Stars, 2021-02
    94/100 stars
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    The dual secreted luciferase L1 retrotransposition assay. (A) Timeline of dual secreted luciferase L1 retrotransposition assay. HeLa cells transfected with 99-PUR-RPS- mGLucI and pSV40-CLuc were exposed to a test compound and puromycin for 2 days. The cells were then exposed to the test compound alone for an additional 3 days. GLuc and CLuc luminescence in the medium and cellular ATP were measured with a luminometer. GLuc luminescence indicates L1 retrotransposition activity. CLuc luminescence was used for normalization as an internal control. Cell viability was evaluated by cellular ATP content. (B) Effect of JM111 (L1 RP ORF1 mutant) on L1 retrotransposition activity. Either 99-PUR-RPS- mGLucI or 99-PUR -JM111- mGLucI containing two missense mutations in L1 RP ORF1 and pSV40-CLuc were co-transfected. L1 retrotransposition activity is represented as normalized luminescence. *** p

    Journal: PLoS ONE

    Article Title: In Vitro Screening for Compounds That Enhance Human L1 Mobilization

    doi: 10.1371/journal.pone.0074629

    Figure Lengend Snippet: The dual secreted luciferase L1 retrotransposition assay. (A) Timeline of dual secreted luciferase L1 retrotransposition assay. HeLa cells transfected with 99-PUR-RPS- mGLucI and pSV40-CLuc were exposed to a test compound and puromycin for 2 days. The cells were then exposed to the test compound alone for an additional 3 days. GLuc and CLuc luminescence in the medium and cellular ATP were measured with a luminometer. GLuc luminescence indicates L1 retrotransposition activity. CLuc luminescence was used for normalization as an internal control. Cell viability was evaluated by cellular ATP content. (B) Effect of JM111 (L1 RP ORF1 mutant) on L1 retrotransposition activity. Either 99-PUR-RPS- mGLucI or 99-PUR -JM111- mGLucI containing two missense mutations in L1 RP ORF1 and pSV40-CLuc were co-transfected. L1 retrotransposition activity is represented as normalized luminescence. *** p

    Article Snippet: The JM111 mutant contains two missense mutations in L1RP ORF1 that abolish retrotransposition . pCMV-GLuc (New England BioLabs) constitutively expresses Gaussia luciferase under a CMV promoter. pSV40-CLuc (New England BioLabs), which contains an SV40 promoter and Cypridina luciferase (CLuc) , was used as an internal control vector for the high-throughput 96-well retrotransposition assay.

    Techniques: Luciferase, Transfection, Activity Assay, Mutagenesis

    Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p

    Journal: Oncotarget

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation

    doi: 10.18632/oncotarget.19108

    Figure Lengend Snippet: Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p

    Article Snippet: Subgenomic replicon plasmids of the Con1 strain (HCV genotype 1b), pFK-I389Luci/NS3-3’/NK5.1 [ ], were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany). pSGR-I389/GLuc was constructed by substitution of GLuc into the Luc gene of pFK-I389Luci/NS3-3’/NK5.1. pSGR-I389/GLuc-GND was constructed by introduction of a mutation in NS5B by PCR using the primers: 5’-CCGTGTTGAGGAGTCAATCTAC-3’(forward) and 5’-GTGTCTAGCTGTCTCCCACG-3’ (reverse). pSGR-JFH1, pHH/SGR-JFH1/GLuc, pHH/SGR-JFH1/GLuc/GND, pcDNA3.1-CD81, pRLucHCVLuc (H77 IRES; HCV genotype 1a), and pRLucEMCVLuc (EMCV IRES) were constructed as previously described [ – ]. pSV40-CLuc was purchased from New England Biolabs. pNL4-3.Luc.R-.E- was obtained from Dr. Niveen Mulholland through the National Institutes of Health AIDS Reagent Program (Bethesda, MD, USA). pNL4-3.NanoLuc.R−.E− was constructed by substitution of NanoLuc (pNL1.1; Promega, Madison, WI, USA) into the Luc gene of pNL4-3.Luc.R-.E-. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was digested with Pme I and Xba I and the fragment was recombined with annealed DNA fragments for miRNA target site and its mutant by the In-Fusion HD cloning kit (Takara, Shiga, Japan).

    Techniques: Transfection, Luciferase, Derivative Assay, Activity Assay, Reporter Assay, Infection, Expressing, Quantitative RT-PCR, Concentration Assay, Cell Viability Assay