Notes: Ebola virus (EBOV) and Reston virus (RESTV) differ in only two amino acid substitutions in the EBOV glycoprotein (GP), however they have distinct host preferences and viral infectivity. Here, these mutations are characterized in a wide range of cell lines and species. The infectivity of viruses is determined with the Steady-Glo® Luciferase Assay. Interestingly, Isoleucine 544/545 is shown to increase infectivity across species. (5174)

Notes: A novel in vivo CRISPER/Cas9-based transcriptional activation (CRISPERa) tool, flySAM, is presented for D. melanogaster. FlySAM is more effective, scalable, and simple allowing for improved overexpression genetic analysis. Activation of transcription was monitored with a luciferase reporter and the Steady-Glo™ Luciferase Assay System. A collection of transgenic TRiP-OE CRISPRa stocks from flySAM is available at https://fgr.hms.harvard.edu/fly-in-vivo-crispr-cas. (5175)

Notes: The authors used a biochemical assay using Xenopus egg extracts to monitor degradation levels of two Wnt pathway components, Axin and ß-catenin, and identify modulators of the Wnt pathway. ß-catenin and Axin were expressed in vitro as firefly and Renilla luciferase fusion proteins, respectively, using the TNT^® SP6 High-Yield Protein Expression System and shown to behave in Xenopus extracts in a similar way to wildtype proteins, which were expressed as radiolabled proteins in rabbit reticulocyte lysate. Degradation of labeled proteins was monitored by SDS polyacrylamide gel electrophoresis and autoradiography, and degradation of luciferase fusion proteins was examined using the Dual-Glo^® Luciferase Assay. Using the Xenopus extract-based assay, the authors screened chemical libraries to identify two modulators of the Wnt pathway, then confirmed this inhibition in HEK 293 cells by demonstrating a decrease in ß-catenin expression with increasing concentrations of the inhibitors. This decrease in ß-catenin-Fluc levels was measured using the Steady-Glo™ Luciferase Assay, and luciferase activity was normalized to cell viability, as determined using the CellTiter-Glo^® Assay. (4181)

Notes: These authors investigated whether HaloTag® technology could be used to specifically target and degrade intracellular proteins. They developed a method to degrade specific proteins of interest using a small molecule by tagging the HaloTag® protein with an adamantyl moiety. The hypothesis was that appending a hydrophobic residue to a protein surface domain would mimic partial denaturation and induce proteasomal degradation. They first used a HaloTag®-luciferase fusion protein to determine the biological activity of various small molecules that enhanced hydrophobicity. After selecting the small molecule that reduced luciferase activity in HEK293 cells, they went on to test their hypothesis in various in vitro and in vivo models. They were able to demonstrate degradation of various HaloTag®-linked transmembrane proteins expressed in HEK293 cells, to show degradations of target proteins in Zebrafish, and to show degradation of the HRAS oncogene product both in NIH3T3 cells and in a mouse tumor model. (4125)

Notes: The authors created two new osteosarcoma models expressing the firefly luciferase enzyme, using a modified pGL3 plasmid to insert luciferase into lentiviral particles for transfection. Luciferase activity was detected using Steady-Glo® Luciferase Assay System with varying numbers of osteosarcoma cells in 96-well microplates. The luciferase-expressing osteosarcomas showed conserved osteolytic and osteogenic activities in mice, and could be imaged in vivo. The authors developed protocols to administer small interfering RNA to its osteosarcoma target (luciferase), and demonstrated its efficiency in vivo to suppress luciferase expression as a model for siRNA treatment of cancer. (4127)

Notes: The Fanconi anemia (FA) pathway is inactivated in a variety of human tumors. Identifying novel compounds that affect FA-pathway deficient cells could provide further information on the FA pathway as well as new therapeutic possibilities. In this study, RKO cells, a human cancer cell line stably expressing firefly luciferase under the control of the p53 consensus DNA-binding site, were treated with 10 and 20µmol/L 80136342, a new compound that enhances cancer cell survival, or 50µmol/L etoposide, a known activator of p53, for 24 hours. The reporter activity was assessed using the Steady-Glo^® Luciferase Assay System and compared to that seen in untreated controls. (3600)

Notes: The authors wanted to screen inhibitory compounds for the HIV-1 accessory protein Nef using both computer modeling and experimental methods. Using a structure-based program for the SH3 binding surface of Nef, drug compounds were screened in silico and then further analyzed using a cell-based assay. The Nef gene and SH3 domain were cloned into the pACT and pBIND Vectors of the CheckMate™ Mammalian Two-Hybrid System, transfected into COS-7 cells, and 18 hours later, the cells exposed to potential inhibitors. After 24 hours, luciferase activity was assessed using either the Dual-Glo™ or the Steady-Glo^® Luciferase Assay Systems. (3751)

Notes: The effector cells of a high-throughput cell-cell fusion assay were dispensed in 384-well plates and expression of the envelope gene induced. Small-molecule compounds or antibodies being tested for their ability to prevent cell fusion were added to the effector cells (carrying firefly luciferase under control of HIV-2 LTR) prior to adding the target cells. The cells were cocultured for 20–24 hours, and luciferase expression was measured using the Steady-Glo^® Luciferase Assay System. (3731)

Notes: Methylation of the WTH3 promoter region has been associated with multidrug resistance in breast cancer cells in vitro. In this study, the effect of WTH3 in primary cultured multidrug resistant cells was evaluated, and the influence of a frequently methylated CpG23 region on WTH3 promoter activity was investigated using a luciferase reporter assay system. Promoter regions containing wildtype or mutated CpG regions were cloned into the pGL3 Vector and transfected into paired MCF7 cell lines, along with a control pGL3 Vector without insert, and a plasmid containing the β-galactosidase gene as a transfection efficiency control. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System, and β-galactosidase activity was determined using the Beta-Glo® Assay System. Luciferase activities of the wildtype and mutant promoter regions were compared after normalization to β-galactosidase activity and protein concentration. (3457)

Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase^® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo^® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells. A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

Notes: HeLa and Saos-2 were transiently transfected with luciferase reporter vectors made from the pGL3-Promoter vector containing various intron sequences from the human integrinα3 gene ITGA3. The first intron sequence was originally cloned by PCR cloning into the pGEM®-T Easy vector. Cell cultures were assessed for luciferase activity by making lysates with the Glo Lysis Buffer and assaying with the Steady-Glo® Assay kit. (3225)

Notes: To test the effect of BMS-378806, a new small molecule inhibitor of HIV-1, a cell fusion assay was developed. Target cells that stably expressed CD4, CXCR4 or CCR5 receptors and carry a responsive luciferase plasmid were prepared.  Effector cells were transiently transfected with HIV coat protein gp160 from various strains of virus, and a plasmid to activate the responsive element promoting luciferase. Therefore, if the cells fuse, luciferase is activated.  To measure this activation, effector cells were plated with target cells in a 1:2 ratio and seeded into 96-well plates at 1.5 x 10⁴ cells/well and incubated with various concentrations of BMS-378806 for 12-24 hours. Luciferase activity was determined with the Steady-Glo^® Reagent. (2737)

Notes: Nuclear receptor ligand binding regions from the Farnesoid-X-activated receptor (FXR) and peroxisome proliferator-activated receptor delta (PPARδ) were fused to a Gal4 DNA Binding Domain. These constructs were used to screen compounds from a LOPAC library of 640 compounds. Ligands bound to the Nuclear receptor ligand binding regions would result in the fusion proteins binding and inducing expression of a luciferase reporter construct with 5 upstream Gal4 binding element repeat sequences. For these experiments, Human hepatoma cell line (Huh7) cells transiently transfected with both plasmids were seeded in 384 well plates. After exposure to the various compounds in the LOPAC library luciferase expression was assessed with the Steady-Glo^® Luciferase Assay System. Cells and Steady-Glo^® reagent were added to cultures by a Labsystem Multidrop-384 liquid dispenser. Plates were read on a Molecular Devices ChemiLuminescence Imaging Plate Reader. (3265)

Notes: To help determine the usefulness of a recombinant Adenovirus for gene delivery and vaccination, the authors compared various methods to determine the amount of anti-Adenovirus serotype 5 (Ad5) neutralizing activity present in human sera. This study describes a system using firefly luciferase in Ad5 vector to infect A549 human lung carcinoma cells.  Using a range of 8,000 to 5 virus particles/cell, the Ad5-luciferase was added to 10⁴ cells/well in a 96-well plate in a total volume of 200µl. After 24 hours, the medium was discarded, and 100µl of PBS was added to each well followed by 100µl Steady-Glo^® Luciferase Assay Reagent. After 15 minutes incubation at room temperature, 100µl from each well was transferred to a black and white isoplate and luminescence was measured on a 1450 Microbeta Trilux. An alternate neutralization assay method used the MTT-based CellTiter 96^® Non-Radioactive Cell Proliferation Assay to score the Ad-mediated cytopathic effect by staining for viable cells and subsequent analysis of optical density. (3095)

Notes: The species-specific response of mouse hepatoma (Hepa.2D) and guinea pig adenocarcinoma (GPC.16) cells to flavonoid treatment was explored in this study. Cell lines stably transfected with dioxin responsive element-driven luciferase constructs were treated with a series of flavanoids. Luciferase expression was measured using the Steady-Glo®> Luciferase Assay system. To increase the number of cells available, cells were grown on Cytodex-1 beads in a larger volume, then split into an opaque 96-well plate prior to application of the Steady-Glo® Reagent. Total RNA was isolated using the SV Total RNA Isolation System, then used in real-time PCR to quantitate luciferase and GAPDH RNA levels after treatment. (3091)

Notes: Researchers stably transfected a vector containing a luciferase reporter gene under the control of Tp53 DNA binding site concatemers into Hs 766T human pancreatic cancer cells. The cells were then plated in 96-well plates and treated with a concentration of 2μg/ml of one of 16,000 individual compounds. Cells were then screened for up-regulation of Tp53-induced luciferase gene expression using the Steady-Glo^® Luciferase Assay System.  Results were analyzed by Wallac Trilux plate reading luminometer. Erratum in: Carcinogenesis. 2003 Oct;24(10):1713. (3195)

Notes: An IL-1 inducible fragment of the murine IL-2 promoter and five tandemly-arranged NFκB binding sites were cloned into the pGL3-Basic Vector, creating the constructs pGL3-IL-2 and pGL3-5xNFκB, respectively. IL2 promoter activation and NFκB activation were measured after rhIL-1α - or PMA-stimulation of EL4D6/76 cells transiently transfected with these constructs. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System. As an internal control, cells were co-transfected with the pRL-SV40 Vector, and expression of firefly and Renilla luciferase was measured using the Dual-Luciferase® Reporter Assay System. (2427)

Notes: These authors used the Steady-Glo^® Luciferase Assay System and Reporter Lysis Buffer in their studies on the 5´ UTR of p27 mRNA. (2434)

Notes: In this study, the Steady-Glo^® Luciferase Assay System was used to monitor luciferase activity in COS-1 and HeLa cells transfected with various reporter constructs. (2436)

Notes: The authors of this study have developed a high throughput method for screening protein-protein interactions in 384-well plates. Using elements of the CheckMate™ Mammalian Two-Hybrid System, mouse cDNAs were linked to the GAL4 DNA binding and VP16 transcription activation domains via a linear cassette PCR strategy. The linear DNAs were mixed with the pG5luc plasmid, transfection reagent and CHO-K1 cells.  Twenty hours after transfection, activation of luciferase by GAL4 binding was assayed using the Steady-Glo^® Luciferase Assay System. Biotec and Tecan liquid handlers were used to semiautomate the system, allowing screening of 20,000 assay wells per day. (2727)

Notes: In this study, a fragment of the nitric oxide synthase type 2 (NOS2) promoter was cloned into the pGL3-Basic Vector. C6 glioma cells stably transfected with this construct were then exposed to various NOS2 inducers and lactacystin. Luciferase activity was measured using the Steady-Glo^® Luciferase Assay System. (2435)