Notes: The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing. (3429)

Notes: These authors analyzed gene expression profiles in the prostate cancer cell line PC3 upon induction of Akt activity to try to identify genes regulated by Akt that participate in the transformation of cells. They identified one mRNA of interest (Fra-1) and cloned its 5' regulatory region into a pGL3-Basic firefly luciferase reporter construct. This construct was used to transiently transfect MCF7 human breast cancer cells along with an Akt plasmid construct and a control vector expressing Renilla luciferase. The firefly construct was induced 4- to 5-fold by co-transfection with Akt3. Transfection conditions were as follows: MCF-7 cells were grown to 70% confluence in six-well plates, then incubated for 15 min with a mixture of 5ng of the control Renilla plasmid, 0.5μg of the Akt-expressing plasmid, and 0.5μg of pFra-luc construct and Fugene® 6 reagent at a 3:1 transfection reagent:DNA ratio. After 48 hours, luciferase activity was assessed using the Dual-Luciferase® Reporter Assay System.

(4361)

Notes: In this paper, the role of microtubules on regulation of hypoxia-inducible factor (HIF) 1-α was explored. A549 human lung cancer cells were transiently transfected with 5µg of NFκB super-repressor plasmid or 3µg of either HA-tagged wild type or mutated HIF-1α construct using FuGENE® 6 Transfection Reagent in 6cm dishes. Twenty-four hours post-transfection, the cells were treated with vinblastine or colchicine before lysing the cells and analyzing the lysate by Western blotting using anti-HIF-1α or anti-HA antibodies (Figures 3 and 6 in article). A549, hepa1c1c7 and hepa1c4 cells were transiently cotransfected with 0.4µg of an iNOS luciferase construct or an NFκB-dependent luciferase vector and 4ng of a CMV Renilla luciferase plasmid in six-well plates. After six hours, the cells were treated with vinblastine, colchicine, nocodazole and paclitaxel and incubated for an additional 6–10 hours. The luciferase activity was then assessed using the Dual-Luciferase® Reporter Assay System (Figures 2 and 4). (4293)

Notes: These authors created a construct encoding a Renilla and firefly luciferase fusion protein to examine the efficiency of site-specific DNA repair. The Renilla gene was taken from the pRL-null Vector and the firefly luciferase gene originated from the pSP-luc+NF Fusion Vector. The fusion construct was created by ligating the C-terminus of the Renilla luciferase gene to the N-terminus of the firefly gene.  The fused proteins were expressed at a constant ratio when transfected into mammalian cells.  Firefly luciferase expression was eliminated by deleting a T at position 213 creating an ochre translational stop codon and placing the downstream sequence out of frame. The mutant protein was then tested for repair using two methods: small fragment homologous replacement and oligonucleotide-mediated repair. The Renilla:firefly expression ratio was tested in several human, murine and simian cell lines and assayed 48 hours post-transfection using the Dual-Luciferase^® Reporter Assay System.  In addition, the repaired plasmids were recovered to verify the sequence correction. (3064)

Notes: In this paper, the authors describe use of ImProm-II™ Reverse Transcriptase to transcribe cDNAs for Quantitative RT-PCR. Reverse transcription (RT) reactions were performed with 1μg total RNA from treated rat PC12 cells. Light Cycler reactions were setup with 1-3μl cDNA from the RT reactions. The researchers incubated cells with EGF and bFGF and analyzed HIF-2α mRNA levels. For these experiments, PC12 cells were incubated with 30ng/ml EGF or 50μM bFGF for 8 hours. Erk1&2 phosphorylation was examined by Western blot using the Anti-ACTIVE® MAPK pAb. Western results were visualized by chemiluminesent detection and analyzed with a digital luminescent image analyzer. The Dual-Luciferase® Reporter Assay System was used to analyze PC12 cell-expressed luciferase from pEpoEm1-luc and pEpoE-luc promoter constructs that were normalized with the pRL-TK vector. Cells were harvested 17-18 hours post transfection and treatment. (2725)

Notes: MCF-7 cells were transfected with a plasmid encoding constitutively active Akt-3. To assess estrogen-related transcriptional activity, the cells were transfected with an ERE-luciferase (firefly) reporter and a Renilla luciferase control plasmid. Dual luciferase assays were performed to determine the effect of estrogen on transcription in these Akt-3 expressing cells. Additionally, proliferation assays were performed using the CellTiter® AQ_ueous One Solution Cell Proliferation Assay and showed that the Akt-3 expression confers serum-independent growth on the cells. (2709)

Notes: Luciferase reporter gene constructs based on the pGL3 vector that contained either MMP-2 or MMP-9 promoters, SV40 promoter (positive control ) or the luciferase basic vectors were transfected into metastatic cells, S53JB-V cells or UMUC-3 cells along with a pβ-actin RL control construct. Cells were treated with an anti-IL-8 antibody, IgG control or no antibody. Dual luciferase reporter assays were performed to determine the effect of the treatments on activity and expression of the MMP-2 and MMP-9 constructs. (2716)

Notes: These researchers used luciferase-containing T-DNA insertions in Arabidopsis thaliana for gene trapping. Luciferase was chosen because its transient expression allowed temporal expression studies. Several insertion vectors were constructed and found to have different insertion frequencies. Vectors containing the luc+ gene had substantially higher insertion rates than native luciferase vectors. Luciferase activity was measured in vivo with a CCD camera or, for longer term studies, with an automated scintillation counter sampling every 15-25 minutes over one week. The application of IRES sites in gene trapping experiments was also investigated using firefly and Renilla luciferases. The Dual-Luciferase® Reporter Assay System was used to monitor luciferase activity in vitro. Finally, to sequence the T-DNA insertion sites, genomic DNA was isolated from T₂ seedlings using the Wizard® Magnetic 96 DNA Plant System and was subsequently amplified and sequenced. (2787)

Notes: The pGL3-basic and pGL3-Promoter vectors were used to make constructs with varying lengths of the mouse LAMA1 promoter. Gene fragments up to 6.1 kb were cloned into the pGL3-promoter vector and analyzed for luciferase expression in a variety of cell lines using the Dual-Luciferase^® Reporter Assay System. F9, NIH/3T3, PYS-2 and EHS cells were transiently transfected with 200ng of reporter construct and 20ng of the Renilla luciferase-expressing phRL-null vector in 24-well plates.  Forty-eight hours later, cell lysates were harvested with Passive Lysis Buffer. Site-directed mutagenesis of  the LAMA1 gene promoter was performed with the GeneEditor™ in vitro Site-Directed Mutagenesis System.  (2631)

Notes: The authors explored the role of beta-catenin and T-cell factor 4 to transactivate vimentin, a protein involved in gain of mesenchymal characteristics and loss of epithelial characteristics as a precursor to metastasis. The vimentin promoter was cloned into the pGL3-Basic Vector. The beta-catenin/TCF binding sites upstream of a minimal c-fos promoter drove firefly luciferase expression in plasmids called TOP-FLASH (wild-type binding sites) and FOP-FLASH (mutant binding sites).  To examine beta-catenin/TCF-4 induction, several human mammary epithelial cell lines were transfected with a mixture of 0.15µg of various firefly reporter constructs (wild type vimentin promoter, mutant vimentin promoter, TOP-FLASH,or FOP-FLASH), 0.15 µg of the beta-catenin expression vector (or the corresponding empty vector), 0.15 µg of the TCF-4 expression vector and 0.8 ng of the Renilla luciferase vector, phRG-TK.  Twenty-four hours after transfection, the cells were lysed and assayed using the Dual-Luciferase^® Reporter Assay System. (3087)

Notes: To investigate how interferon gamma stimulates expression of the murine Clara cell secretory protein (CCSP) gene, the 280bp CCSP promoter region was radiolabeled and incubated with nuclear extract from mouse transformed Clara cells (mtCC). Transcription factor binding sites were identified using the Core Footprinting System. A 30bp section of the CCSP promoter containing three transcription factor consensus sites was synthesized with 0, 1 or 2 mutated binding sites and tested in the presence of mtCC nuclear extract. Oligos and nuclear extract were allowed to form complexes with or without interferon gamma in Gel Shift Binding Buffer. A 166bp section of the CCSP promoter was also cloned into the pGL3-Basic Vector and co-transfected with the pRL-TK Vector into mtCC. The cells were subjected to interferon gamma treatment and the cell lysates assayed using the Dual-Luciferase^® Reporter Assay System. (3112)

Notes: Researchers created promoter constructs in the pGL3-Basic vector to study NHE3 promoter function in cotransfection experiments with the pRL-null vector.  Transfected Caco-2 cells were analyzed by the Dual-Luciferase^® Reporter Assay System. Beta-Galactosidase Assays were also performed on SL2 cells cotransfected with the pRL-null vector. The Renilla Luciferase Assay System was used to normalize these transfectants. (2642)

Notes: Researchers used luciferase reporter constructs along with pRLSV40 or phRL-null Vectors in transiently transfected NIH3T3, CHO, 293, HeLa, and Rat chondrosarcoma cell lines.  Transfections were performed in 6-well plates. Lysates of the transfectants were prepared at various times post-transfection and were analyzed with the Dual-Luciferase^® Reporter Assay System.  (2726)

Notes: Mig-6 cDNA was cloned into the pSI Vector to be used in cotransfection experiments with luciferase reporter constructs.  The phRL-CMV Vector was added to the transfections to normalize the data generated by the Dual-Luciferase^® Reporter Assay System in NIH/3T3 cells. (2632)

Notes: The promoter region of human CYP19 was amplified from a lambda library and cloned into the pGEM^®-T Easy Vector for sequencing. Several mutants of this aromase cytochrome P450 promoter were created and cloned into the pGL3-Basic Vector. One microgram of the various reporter constructs along with 25ng pRL-null Vector (as an internal control) were transfected into HMEC-1 and MCF-7 cells in six-well plates. Luciferase levels were assayed 48 hours post-transfection using the Dual-Luciferase^® Reporter Assay System. To further characterize the CYP19 promoter, a 30-mer region with a GATA motif was added to 250ng HMEC-1 nuclear extract in the presence of oligonucleotide competitors or antibodies using the Gel Shift Binding 5X Buffer, then analyzed by gel electrophoresis. (3110)

Notes: BE(2)-M17 and HEK-293T cells (Human dopaminergic neuroblastoma and human embryonic kidney cells, respectively) were cultured in Opti-MEM (Life Technologies) with 10% FBS, penicillin (100 units/ml) and streptomycin (100 mg/ml). Cells were plated at 80% confluence and maintained in an atmosphere of 5% CO₂ at 37°C for twenty-four hours prior to transfection in 24-well culture plates. Firefly Luciferase-containing constructs (pGL3) were co-transfected with the phRL-TK synthetic Renilla luciferase vector, at a molar ratio of 1:100 (phRL-TK:pGL3).  These transfections were performed in serum-free media for 12 h using a 1:3 molar ratio of DNA: Fugene reagent (Roche Biochemicals), and 0.2 mg of DNA per well. After forty hours cells were rinsed with PBS and then harvested with Passive Lysis Buffer. The Dual-Luciferase^® Reporter Assay System was used to measure firefly and Renilla luciferase activities from the transfected cells using duplicate readings on a Turner Designs 20/20 Single-Injector Luminometer. (2663)

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: The researchers used pGL3 basic constructs and the phRL-TK vector in cotransfection studies to examine the effect of hypoxia on promoter regions of DEC1 and DEC2 promoter constructs in ATDC5, 293T, and HeLa cells. The Dual-Luciferase^® Assay System was used to examine the expression levels of luciferase from the constructs. (2630)

Notes: In this paper, total RNA isolated from transgenic mouse pre-B cells was used as template for semi-quantitative RT-PCR. The RNA was isolated using the RNAgents^® Total RNA Isolation System. Promoter studies were performed in 293T cells with the Dual-Luciferase^® Reporter Assay System. Firefly luciferase activity controlled by Renilla luciferase activity was provided by the pRL-TK Vector. (2566)

Notes: To study response to the thyroid hormone (T₃), a firefly luciferase construct was made with a T₃ responsive element upstream of a thymidine kinase minimal promoter. This plasmid and the Renilla luciferase reporter plasmid phRL-SV40 were injected into the dorsal muscle of NF55 stage Xenopus tadpoles. One microliter of solution containing between 0.7 and 2.1μg of DNA in 0.07M NaCl with a tracking dye were injected. After two days, the dorsal muscles were collected, flash frozen and sonicated in Passive Lysis Buffer. This homogenate was assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System. (2762)

Notes: The authors examined how the transcription factor AP-2 interacts with and is affected by UBC9, a SUMO-conjugating enzyme. Promega's GeneEditor™ in vitro Site-Directed Mutagenesis System was used to create a single amino acid change (K10R) in AP-2γ once it was determined that the lysine was required for sumolation in vivo. Transient transfection assays were performed using the TransFast™ Transfection Reagent. All cell lines (MDA MB 436, MCF7, T47D and HepG2) were transfected in 12-well plates when 60-70% confluent. Various amounts of plasmid were used during transfection and detailed in the paper. For reporter genes, the AP-2 firefly reporter (3xAP2-Bluc) and Promega's pRL-TK Vector were used in a 1:1 ratio, the Renilla luciferase used to normalize firefly expression.  Lysates were made 48 hours post-transfection and assayed using the Dual-Luciferase^® Reporter Assay System. (2558)

Notes: In this study, the phRG-B Vector was used as an internal control to monitor transfection, and the Dual-Luciferase^® Reporter Assay System was used to measure luciferase activity of control and experimental reporters. (2425)

Notes: Tumore necrosis factor α-induced apoptosis of permanently transfected HeLa cells expressing the IG20 cDNA or its splice isoforms were assayed using the CaspACE™ FITC-VAD-FMK In Situ Marker. The activation os specific caspases was determined using the CaspACE™ Assay System. The Dual-Luciferase Assay System was used to measure TNF α-induced NF-κB activation in HeLA-IG20 cells. (2418)

Notes: NIH3T3 and HEK293-T cells were transfected with different expression plasmids together with 0.1 µg of each reporter plasmid and 0.01 µg of pRL-null plasmid as an internal control. Cells were grown in 6 well plates. (2136)

Notes: The ability of a 5' UTR to act as an internal ribosome entry site (IRES) was explored in this study. The 5' UTR of YAP1 and p150 was cloned between the Renilla and firefly luciferase genes driven by a GAL1 promoter. The IRES effect was examined by measuring firefly luciferase expression driven by the IRES normalized against the first cistron, Renilla luciferase. To use the Dual-Luciferase^® Assay in yeast, the liquid culture was pelleted then resuspended in Passive Lysis Buffer. Glass beads were added to the mixture, which was then and vortexed using two, 30-second pulses. This mix was then harvested at high speed and 20μl of the supernatant was used in the Dual-Luciferase^®Assay. (3032)