Notes: Three plasmids were transfected into M12 cells or Jurkat cells: Cytokine promoter-driven firefly luciferase reporter vector; GATA-3 expression vector and pRL-CMV Vector. The vectors were electroporated at a 40:40:1 ratio. Luciferase activity was determined with the Dual-Luciferase^® Reporter Assay System. (0497)

Notes: Reporter studies were performed in K562 cells. The Dual-Luciferase^® Reporter Assay System was used to measure the firefly luciferase from a GAL4 binding site-containing luciferase vector and cotransformed pRL-SV40 Vector. No ratios were reported. Other vectors were transfected as well and produced proteins that activated that bound the GAL4 domain and activated transcription of the firefly luciferase in a two-hybrid-type assay. Another vector was constructed with Heat Shock Elements instead of GAL4 domains in a pGL3 Promoter Vector. (0116)

Notes: Promoter studies were performed in HeLa cells. The promoter constructs were assembled in the pGL3 Basic Vector (10µg) and cotransformed with the pRL-SV40 Vector (0.1µg) at a 10:1 ratio using the calcium phosphate method. Reporter activity was measured with the Dual-Luciferase^® Reporter Assay System. The TNT^® T7 Quick Coupled Transcription/Translation System was used to produce a positive control protein for Western blot analysis of transfected HeLa cell extracts. (0115)

Notes: The authors cloned a 2.6kb promoter-enhancer region of the MHC IIB gene into the pGL3-Basic Vector. They injected this construct along with the pRL-CMV Vector into mouse muscle cells. Tissue extracts were prepared with a buffered Tris solution containing a protease inhibitor cocktail and assayed for reporter activity using the Dual-Luciferase^® Reporter Assay System. (0287)

Notes: Luciferase studies were performed in a Rat-1 fibroblast cell line with an inducible Ha-ras gene. Experimental pGL3 Basic vectors were cotransfected with pRL-TK at a 2:1 ratio. (0389)

Notes: Studies were performed in F-36P, a human IL-3 dependent erythroleukemia cell line. Cells were electroporated with either an AP-1 specific luciferase reporter vector or a STAT5-specific luciferase reporter vector. To normalize for electroporation efficiency, an equal amount of the pRL-CMV Vector was co-transfected with the luciferase reporters. Luciferase activities were monitored with the Dual-Luciferase® Reporter Assay System. (0599)

Notes: Studies were performed in HepG2 cells. Cells were transfected with a hepatocyte nuclear factor-6 (HNF-6)–responsive promoter firefly luciferase construct (3µg), various forms of the HNF-6 protein in an expression vector (400ng) and a Renilla luciferase control vector (pRL-null Vector) driven by the liver pfk-2 promoter (not responsive to HNF-6; 500ng). The ability of the various HNF-6 isoforms to drive luciferase expression was normalized to Renilla luciferase activity, and the luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. The various HNF-6 constructs were also expressed in vitro with the TNT^® Coupled Wheat Germ Extract System and used for gel shift assays. (0839)

Notes: Promega's Taq DNA polymerase was used in construction of various plasmids. The authors used a mammalian two-hybrid system with a firefly luciferase reporter and pRL-CMV Vector as a transfection control. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. (1266)

Notes: Reporter studies were performed in the ME-180 squamous cell carcinoma cell line. One microgram of the firefly luciferase construct was contransfected with 1ng of pRL-CMV Vector (1,000:1 ratio of firefly reporter to Renilla control), and luciferase activities were measured with the Dual-Luciferase^® Reporter Assay System. The Kanamycin Control RNA was used as a control template for primer extension analysis. (1065)

Notes: The authors used the Dual-Luciferase^® Reporter System with U937 cells and the pRL-TK Vector. (0977)

Notes: Studies were performed in HepG2 and HuH7 cells (both human cells of hepatic origin). One microgram of the experimental constructs in the pGL3 Basic Vector was cotransfected with 50ng of the pRL-SV Vector (20:1 ratio). Luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System. (1443)

Notes: The 293 cell line was cotransfected with a luciferase reporter containing the IL-2 promoter, an expression vector for either EGR-1, NFATc or both and finally the pRL-TK Vector. Reporter to control ratio was 4:1 and the luciferase activity was determined with the Dual-Luciferase® Reporter Assay System. (1265)

Notes: Reporter assays were performed in primary murine splenocytes. The interferon-gamma promoter constructs were assembled in the pGL3-Basic Vector and a Renilla luciferase control vector was constructed with the pRL-null Vector and a β-actin promoter. The plasmids were electroporated at a 10:1 ratio. Luciferase activities were followed with the Dual-Luciferase^® Reporter Assay System. (0285)

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 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)