Notes: Studies were performed in HepG2 cells. Experimental promoter constructs were assembled in the pGL3-Basic Vector and transfection efficiency was monitored by cotransfecting the pRL-CMV Vector. Luciferase activities were assayed with the Dual-Luciferase® Reporter Assay System. The ratio of experimental:control vector were not reported. (0219)

Notes: The authors used the PolyATtract® mRNA Isolation System to isolate mRNA from total RNA. They used this mRNA-rich fraction for primer extension analysis using AMV Reverse Transcriptase. The Wizard® Plus Midipreps DNA Purification System was used for various plasmid isolations. The Dual-Luciferase® Reporter Assay System was used to study promoters cloned into the pGL3-Basic Vector. The pRL-SV40 was used as a transfection control plasmid. (0095)

Notes: Studies were performed in HepG2 cells and mouse C2 skeletal muscle cells. The experimental pGL3-Basic Vector-derived constructs were cotransfected with pRL-CMV Vector using the calcium phosphate method. Luciferase activity was monitored with the Dual-Luciferase^® Reporter Assay System 48 hours after transfection. (0899)

Notes: The authors used the Dual-Luciferase^® Reporter Assay System to study IRES sequences. They placed the Renilla gene upstream of the firefly gene with sequences containing and IRES sequence between them. Then they performed deletional studies on this region to identify minimal necessary sequences for IRES function and allowed for both firefly and Renilla luciferase expression. (1032)

Notes: The authors cloned promoter region (and deletion mutants) of mouse ribonucleotide reductase R1 gene into pGL3-Basic Vector. Also, they cloned 3´-UTR of this gene downstream of firefly luciferase coding region in pGL3-Control Vector to study posttranscriptional effects mediated by this region. Co-transfected plasmids with pRL-SV40 into Balb/3T3 fibroblasts. Assayed luciferase activity with Dual-Luciferase^® Reporter Assay System. (0985)

Notes: Dual-Luciferase^® Reporter Assay System was used to perform studies in rat PC12 cells. The firefly luciferase reporter vector was transfected with the control plasmid (pRL-TK Vector) at a 20:1 ratio. Eighteen hours after transfection, the cells were treated with NGF, EGF or KCl and assayed up to eight hours later. In some experiments, PC12 cells were co-transfected with the firefly luciferase plasmid, the Renilla control vector and a vector expressing the wildtype or mutant NGFI-B protein. (0920)

Notes: Promoter activity was assayed in JEG-3 choriocarcinoma cells, JAR choriocarcinoma cells, HeLa cells and primary adipocytes. The pGL3 Vectors were transfected at a 20:1 ratio over pRL-CMV Vector. Enhancer activity was functional only in the choriocarcinoma cells. The Dual-Luciferase^® Reporter Assay System was used to measure luciferase expression. (1446)

Notes: This paper describes a method for quantitation of luciferase mRNA by in vitro translation using Promega TNT^® Coupled Wheat Germ Extract System. (Rabbit reticulocyte lysate could not be used because of luciferase quenching problems). In this reporter gene assay, COS cells were transfected with a firefly luciferase reporter plasmid driven by promoters/enhancers of varying strengths and total cellular RNA was isolated and translated in vitro using the TNT^® System. To normalize expression, a CMV Renilla luciferase or beta-galactosidase vector was used as a control. To measure luciferase activity either Promega Luciferase Assay System or Dual-Luciferase^® Reporter Assay System was used. (2047)

Notes: Studies were performed in H4IIE rat hepatoma cells. The pGL3-based Vectors were co-transfected with the Renilla control vector (pRL-SV40 Vector) at a 6:1 ratio and assayed using the Dual-Luciferase^® Reporter Assay System. (1489)

Notes: The Dual-Luciferase^® Reporter Assay System was used to study transfections of IMR32 and NIH3T3 cells with firefly luciferase vector (pGL3 Basic) constructs. Transfections were controlled with co-transfected pRL-CMV Vector. Tth DNA Polymerase was used as a high temperature reverse transcriptase. SP6 RNA Polymerase was used to produce probes for RNase protection assays. (0558)

Notes: Reporter studies were performed in THP-1 monocytic cells, Jurkat T-cell leukemic cells and K562 myelogenous leukemia cells. Experimental promoter constructs were prepared in the pGL3 Basic Vector. Transfections were control by cotransfection with the pRL-CMV Vector, a source of Renilla luciferase. The activity of the two luciferases was determined with the Dual-Luciferase® Reporter Assay System. Ratios of pGL3-based Vectors to the pRL-CMV Vector were not reported but 0.5µg of the pRL-CMV Vector was used in each transfection. (0630)

Notes: The Dual-Luciferase™ Reporter System was used to quantitate the presenilin promoter activity in Neuro2a neuroblastoma cells, mouse P19 embryonal carcinoma cells and NIH 3T3 cells. Studies were also performed in P19 cells treated with retinoic acid to acquire a neuron-like phenotype and P19 cells treated with dimethyl sulfoxide to acquire a muscle-like phenotype. The presenilin promoter functioned best in the Neuro2a and neuron-like P19 cells. 5´-RACE products from mouse brain RNA were purified with the Wizard^® PCR Preps System and cloned into the pGEM-T Vector . The cloned amplimers were sequenced and used as a template for amplification to produce truncation mutants to assess promoter activity. (1590)

Notes: A firefly luciferase reporter vector containing three copies of the Ig kappa NF-kB binding site was cotransfected with vectors expressing either TRAF2 with or without MAPK Kinase 4 or mutants of TRAF2. All firefly luciferase activity was normalized to Renilla luciferase activity provided by the pRL-TK Vector. These studies were performed in COS 7 cells. Luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. (0508)

Notes: CHO-K1 cells were transfected at ~80% confluency with four plasmids: (1) a CMV-driven vector expressing the wildtype or mutant human insulin receptor; (2) a pGL3-Basic-derived Vector containing five GAL4 binding sites upstream of the firefly luciferase reporter; (3) the pRL-CMV Control Vector; and (4) a fusion of the GAL4-binding domain and Elk-1 transcription factor. The Dual-Luciferase^® Reporter Assay System was used to confirm that specific mutations in the insulin receptor negate its ability to activate the Elk-1 transcription factor. (0896)

Notes: Vascular smooth muscle cells were transfected with a firefly luciferase reporter vector and pRL-SV40 as a control. Transfection efficiency was normalized to the Renilla luciferase activity as determined by the Dual-Luciferase^® Reporter Assay System. (0555)