psv40 cluc  (New England Biolabs)

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

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