Notes: The authors used the Dual-Luciferase^® Reporter Assay System, pGL3 Control Vector, pRL-CMV Vector, pSV-beta-Galactosidase Control Vector, Luciferase Assay System and RNase ONE^® Ribonuclease in their studies. The objective of the paper was to demonstrate that the 5' untranslated region (UTR) of c-myc functions as an internal ribosome entry site (IRES). To directly assay the ability of the UTR to function as a IRES, the Renilla luciferase gene was subcloned in front of the UTR. Dual expression of the luciferases was demonstrated in both HeLa cells and HepG2 cells and dependent upon the c-myc UTR. Mutation of the Renilla luciferase sequence to contain a hairpin structure, demonstrate that the Renilla luciferase activity could reduced by 75% with no effect on the firefly luciferase activity. RNase protection assays with a 624 nucleotide probe that crossed the Renilla luciferase cDNA, the c-myc UTR and the firefly luciferase cDNA demonstrated that only one single message is produced by the transfected HeLa cells and the c-myc UTR is not functioning as a promoter. All transfections were normalized to control β-galactosidase activity. (0339)

Notes: Luciferase studies were performed in the human B cell line, CL-01. Cells were electroporated with a 40µl DNA-TE buffer solution containing 20µg of constructs in either pGL3 Basic or pGL3 Promoter Vectors and 10ng of the pRL-CMV Vector. Thus, a 1:2000 ratio of firefly to Renilla luciferase vector is achieved and activity was measured with the Dual-Luciferase® Reporter Assay System. (1353)

Notes: Luciferase reporter constructs were cotransfected into 3T3-L1 preadipocytes with the pRL-CMV Vector at a 25:1 ratio and luciferase activities were determined with the Dual-Luciferase^® Reporter Assay System. RT-PCR was accomplished with AMV Reverse Transcriptase and Taq DNA Polymerase. RNase Protection Assay were performed with the aid of the RNase ONE™ Ribonuclease. (0988)

Notes: Inserted promoter region and its deletion variants into pGL3-Basic Vector and co-transfected with pRL-CMV Vector (no ratio given). Measured luciferase activities with Dual-Luciferase^® Reporter Assay System. (0952)

Notes: The authors performed DNaseI footprinting using the Core Footprinting System and nuclear extract or recombinant human AP-1 (c-jun). Promoter and enhancer elements of the human β4 integrin gene were analyzed using the Dual-Luciferase^® Reporter Assay System. pRL-TK Vector was used as a transfection control. The transfection ratio of pGL3-Basic-derived plasmids to pRL-TK Vector was 10:1. (0296)

Notes: Luciferase reporter plasmids were cotransfected with pRL-TK Vector at a 5:1 ratio in CPII mouse mast cells. In studies with the reporter vector, ras expression vector and pRL-TK Vector, the ratio of each was 3:5:1 with the amount of pRL-TK Vector constant in all transfections at 2µg per reaction. All transfections were accomplished by electroporation and both luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. (1288)

Notes: Used Dual-Luciferase^® Reporter Assay System with a 400:1 transfection ratio of the firefly luciferase plasmid to pRL-SV40 Vector. Also used the pCI-neo Mammalian Expression Vector to make several E1A expression plasmids. (0743)

Notes: Promoter activities were studied in HepG2 cells with the Dual-Luciferase^® Reporter Assay System. The Renilla control vector was constructed from the pRL-SV Vector by replacing the SV40 promoter with the α-globin gene proximal promoter (–79-+1). The Firefly luciferase construct was prepared in the pGL3 Basic Vector. The pGL3-based Vector and pRL-based Vector were transfected at a 3:1 ratio. A mutant nuclear factor 1 site was introduced into the 1A1 promoter with the GeneEditor™ in vitro Site-Directed Mutagenesis System by converting GCCA to CGCA. (0659)

Notes: Studies were performed in ts13 cells that have a temperature-sensitive TAFII250. Either cyclin A or cdc2 luciferase vectors were cotransfected with wildtype or mutant TAFII250 proteins as well as pRL-CMV Vector (luciferase vector: Renilla vector; 20:1). Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System (0586)

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 assays were performed in the odontoblast cell line, MO6-G3. Experimental promoter constructs were assembled in the pGL3 Basic Vector and transfections were control through the use of the pRL-SV Vector. Luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. Primer extension analysis of the gene transcript was performed with the Primer Extension System. (1196)

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: Human diploid embryonic lung fibroblasts (LU) were transiently transfected using Tfx™-50 Reagent. The cells were 70-80% confluent, and the lipid to DNA ratio was 3:1 (transfection time = 2 hours; no serum). Activation of the cyclin promoter was measured using the Dual-Luciferase^® Reporter Assay System. The Core Footprinting System was used to determine the promoter region that is bound by the cytomegalovirus immediate early protein. (1403)

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: A plasmid, p35S-Rluc, consisting of the CaMV 35S promoter and ADH1-Intron1 fused to the Renilla luciferase gene as a transformation control in maize. The Dual-Luciferase^® Reporter Assay System was used to detect and quantitate expression levels. (0422)

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)