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Promega Corporation

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J. Biol. Chem. 277, 16456-16463. Identification of essential regions in the cytoplasmic tail of interleukin-1 receptor accessory protein critical for interleukin-1 signaling. 2002

Radons, J., Gabler, S., Wesche, H., Korherr, C., Hofmeister, R. and Falk, W.

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)

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J. Biol. Chem. 277, 47014-47021. Identification of Functional Hypoxia Response Elements in the Promoter Region of the DEC1 and DEC2 Genes. 2002

Miyazaki, K., Kawamoto, T., Tanimoto, K., Nishiyama, M., Honda, H., and Kato, Y.

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)

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Mol. Cell. Biol. 22, 5563-5574. NF-kB1 can inhibit v-Abl-induced lymphoid transformation by functioning as a negative regulator of cyclin D1 expression. 2002

Nakamura, Y., Grumont, R.J., and Gerondakis, S.

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)

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Mol. Cell. Biol. 22(24), 8527-8538. Nuclear receptor corepressor recruitment by unliganded thyroid hormone receptor in gene repression during Xenopus laevis development. 2002

Sachs L.M., Jones, P.L., Havis, E., Rouse, N., Demeneix, B.A. and Shi, Y.B.

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 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)

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J. Biol. Chem. 277, 30798-30804. Transcription factor AP-2 interacts with the SUMO-conjugating enzyme UBC9 and is sumolated in vivo. 2002

Eloranta, J. J., and Hurst, H.C.

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)

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Cell 109, 47-60. Wnt/Wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear β-catenin-TCF complex. 2002

Kramps, T., Peter, O., Brunner, E., Nellen, D., Froesch, B., Chatterjee, S., Murone, M., Zullig, S. and Basler, K.

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)

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J. Biol. Chem. 276, 47202. Contrasting effects of IG20 and its splice isoforms, MADD and DENN-SV, on tumor necrosis factor α-induced apoptosis and activation of Caspase-8 and -3 2001

Al-Zoubi, A.M., Efimova, E.V., Kaithamann, S., Martinez, O., El-Azami El-Idrissi, M., Dogan, R.E., and Prabhakar, B.S.

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)

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Nature 411, 494-498. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. 2001

Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T.

Notes: In this landmark paper describing RNA interference in mammalian cells, the firefly and Renilla luciferase gene products were targeted for degradation. NIH/3T3, HEK293, HeLa S3, COS-7, and S2 cells were transfected with 1μg pGL2-Control or pGL3-Control Vector, 0.1μg pRL-TK Vector, and 0.21μg siRNA duplex targeting either firefly of Renilla luciferase. The Dual-Luciferase® Assay was used 20 hours post-transfection to monitor luciferase expression. It was found that transfection with 21bp dsRNA can cause specific degradation of a targeted sequence. This was the first demonstration of the RNAi effect in mammalian cells. (3027)

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Genes Dev. 15, 535-553. Regulation of gene expression by the small GTPase Rho through the ERK6 (p38 gamma) MAP kinase pathway. 2001

Marinissen, M.J., Chiariello, M. and Gutkind, J.S.

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)

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Proc. Natl. Acad. Sci. USA 98(4), 1531-1536. Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells. 2001

Zhou, W., Edelman, G.M., and Mauro, V.P.

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)

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J. Virol. 74, 5776–87. The long terminal repeat of Jaagsiekte sheep retrovirus is preferentially active in differentiated epithelial cells of the lungs. 2000

Palmarini, M., Datta, S., Omid, R., Murgia, C. and Fan. H.

Notes: In general, transient transfections were performed using 2 × 105 to 4 × 105 cells plated in 6-well plates 24 hours before transfection, with a total of 2µg of plasmid (1µg of reporter vector and 1µg of control) using FuGENE® 6 reagent. The reporter vectors were based on the pGL3-Basic and pGL3-Promoter Vectors.

For MLE-15 (mouse lung), mtCC1-2 (mouse Clara cell), NIH-3T3 (mouse embryo), TCMK (mouse kidney), ST3, (mouse thymus) CP-MRI (sheep choroid plexus) and CP-ATCC (sheep choroid plexus) transfections used 0.5µg of reporter plasmid and 0.5µg of pRL-TK or 50ng pRL-null Vector. Reporter plasmid activity was assessed using the Dual-Luciferase® Assay System. (4271)

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Am. J. Hum. Genet. 66, 187-195. Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to emphysema. 2000

Yamada, N. , Yamaya, M. , Okinaga, S. , Nakayama, K. , Sekizawa, K. , Shibahara, S. , Sasaki, H.

Notes: To explore the regulatory effect of a (GT)n repeat on HO-1 gene expression in either A549 cells or Hep3B cells, HO-1 promoter/luciferase fusion genes were constructed using pGL3-Basic Vector and co-transfected with the Renilla luciferase expression vector, pRL-TK Vector. Reporter activities were determined with the Dual-Luciferase® Reporter Assay System. (0130)

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Mol. Cell. Biol. 20(14), 5216-5226. Modulation of CRX transactivation activity by phosducin isoforms 2000

Zhu, X. and Craft, C.M.

Notes: The pRL-null Vector was used as a reporter for a DLR® assay (Dual-Luciferase® Reporter Assay System). The promoter from human IRBP (interphotoreceptor retinoid binding protein) was cloned into the HindIII site of pRL-null. Transient transfections were done with 1µg of the pIRBP, 0.4µg of expression construct, and 0.2µg of pGL3-promoter as a transfection efficiency control. Activity of the Renilla luciferase was normalized to the activity of the firefly luciferase. Cells used for this assay were COS-7 and Weri-Rb-1 retinoblastoma cells. (2149)

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Mol. Cell. Biol. 20(10), 3558-3567. Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression. 2000

Mothe-Satney, I., Yang, D., Fadden, P., Haystead, T.A. and Lawrence, J.C., Jr

Notes: The authors designed a bicistronic reporter, designated pRLIRESFL, encoding Renilla luciferase and firefly luciferase. This construct was used to investigate cap-dependent mRNA translation. pRLIRESFL directs the synthesis of an mRNA from which Renilla luciferase is translated in a manner dependent on the 5' cap and firefly luciferase is translated in a cap-independent manner through the IRES. The Renilla luciferase coding fragment was inserted into the EcoR I cloning site of p2332, a plasmid containing the CMV promoter, sequences encoding the IRES from encephalomyocarditis virus, firefly luciferase and the SV40 t-intron and polyadenylation signal. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. (2173)

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Mol. Cell. Biol. 20(14), 4990-94. Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. 2000

Wilson, J.E., Powell, M.J., Hoover, S.E. and Sarnow, P.

Notes: Control regions of a naturally occurring dicistronic promoter from Cricket Paralysis Virus (CrPV) were cloned into a previously described dicistronic reporter vector coding for both Fluc and Rluc. The reporter was used either for in vitro translation from RiboMAX™-produced uncapped RNA (RiboMAX™ Large-Scale RNA Production System) using either an RRL or WGE system, and measuring the translation using the Dual-Luciferase® Reporter Assay System, or for transfection into SL-2 cells. RNA was also used for transfection of the SL2 cells, and luciferase activities were measured using DLR. Luciferase readings are reported as ratio of Fluc activity to Rluc activity, with the no insert vector ratio set to 1. (2147)

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J. Biol. Chem. 275, 8600-8609.. Oxidative stress disrupts glucocorticoid hormone-dependent transcription of the amiloride-sensitive epithelial sodium channel alpha-subunit in lung epithelial cells through ERK-dependnent and thioredoxin-sensitive pathways. 2000

Wang, H.-C. , Zentner, M.D., Deng, H.-T., Kim, K.-J., Wu, R., Yang, P.-C., Ann, D.K.

Notes: Addition of exogenous H2O2 to cultured A549 cells causes a rapid increase in activated Erk as judged by the Anti-ACTIVE® MAPK pAb via Western blotting. There is an inhibition of transcriptional activation of the amiloride-sensitive epithelial sodium channel α-subunit. The inhibition is reversed by 50µM U0126 MEK Inhibitor treatment 20 minutes prior to addition of H2O2 . Reporters were used for measurement of transcriptional activity and were produced in the pGL2-Promoter Vector. The reporter vectors were cotransfected into the A549 cells with the pRL-TK Vector at a 12:1 ratio. The luciferase activities were measured with the Dual-Luciferase® Reporter Assay System. Transfections were accomplished with the ProFection® Mammalian Transfection System-DEAE-Dextran. (0200)

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Blood Cells Mol. Dis. 25(14), 193-209. Activation of the delta-globin gene by the beta-globin gene CACCC motif. 1999

Ristaldi M.S., Casula S., Porcu S., Marongiu M.F., Pirastu M. and Cao, A.

Notes: These authors created mutations in the delta-globin promoter to incorporate various combinations of three elements present in the beta-globin promoter. They then examined compared the function of this chimeric promoter in Cos7, C88 mouse erythroleukemia (MEL) and K562 erythroid cell lines compared to that of the delta-globin wild type promoter.  The chimeric globin promoters drove expression of firefly luciferase. The pRL-CMV Vector was used as a transfection control at a 3:1 ratio (4 µg total).  Relative expression levels were assayed using the Dual-Luciferase® Reporter Assay System.  The authors also used a competitive transient expression assay using a single vector containing both firefly and Renilla luciferase genes under the control of separate promoters.  Again, the chimeric delta-globin promoter (driving firefly luciferase) was tested with the wild type beta-globin promoter (driving Renilla luciferase). The ratio between the two was expressed relative to expression from a wild type beta-globin promoter driving both firefly and Renilla luciferase in the same construct.  (3059)

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J. Biol. Chem. 274, 8813-8822. CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulated phosphoenolpyruvate carboxykinase (GTP) gene transcription. 1999

Leahy, P., Crawford, D.R., Grossman, G., Gronostajski, R.M., Hanson, R.W.

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to demonstrate a direct interaction between the transactivation domain of nuclear factor I-C (NFIC) and the CREB binding domain of the CREB-binding protein (CBP). Four different domains of the CBP were linked to the GAL4 binding domain of the pBIND Vector, and the transactivation region of the NFIC was cloned into the pACT Vector. The positive control vectors included with the system, pBIND-Id and pACT-myo Vectors, induced a 110-fold increase in luciferase activity. One construct of the CBP protein in combination with the NFIC construct induced a 70-fold increase in luciferase activity. Equal amounts of the vectors (0.6µg) were transfected into HepG2 cells. The Dual-Luciferase® Reporter Assay System was used to assess luciferase activities. (0851)

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J. Biol. Chem. 274, 2672-2681. Cross-talk between janus kinase-signal transducer and activator of transcription (JAK-STAT) and peroxisome proliferator-activated receptor- alpha (PPARalpha) signaling pathways. Growth hormone inhibition of pparalpha transcriptional activity mediated by stat5b. 1999

Zhou, Y. C. , Waxman, D. J.

Notes: The authors co-transfected COS-1 cells with a firefly luciferase reporter construct containing three tandem repeats of a PPRE as well as expression plasmids for growth hormone receptor, JAK2, STAT5a, and PPAR alpha. The pRL-TK Vector was used as a transfection control. Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. (0061)

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J. Biol. Chem. 274, 3522-3530. Distinct and common pathways in the regulation of insulin-like growth factor-1 receptor gene expression by angiotensin II and basic fibroblast growth factor. 1999

Scheidegger, K.J., Du, J., Delafontaine, P.

Notes: The authors co-transfected firefly luciferase reporter constructs with pRL-TK Vector at a ratio of 200:1 into CHO cells overexpressing the angiotensin II AT-1A receptor. Luciferase activities were assayed with the Dual-Luciferase® Reporter Assay System. (0450)

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Oncogene 18, 1-8. Endothelial receptor tyrosine kinases activate the STAT signaling pathway: Mutant Tie-2 causing venous malformations signals a distinct STAT activation response. 1999

Korpelainen, E.I., Kärkkäinen, M., Gunji, Y., Vikkula, M., Alitalo, K.

Notes: The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to produce the R849W mutation in the Tie-2 cDNA cloned into the pcDNA3.1Z+ Vector. The Dual-Luciferase® Reporter Assay System was used with the 293T cells, but no details of the transfection ratio for the firefly luciferase reporter construct and the pRL-TK Vector are provided. (0883)

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J. Immunol. 162, 4069-4078. Expression of murine IL-12 is regulated by translational control of the p35 subunit. 1999

Babik, J.M., Adams, E., Tone, Y., Fairchild, P.J., Tone, M. and Waldmann, H.

Notes: The Dual-Luciferase® Reporter Assay System was used to study a promoter as well as the 5' UTR of the p35 gene. Reporter studies were performed in the pGL3 Enhancer Vector using a 5:1 ratio of the firefly luciferase vector to the pRL-TK Vector. Studies of the 5' UTR were performed in the pGL3 Control Vector, which was transfected at a 10:1 ratio with the pRL-TK Vector. Studies were performed in A20 B cell lymphoma cell line and the RAW 264 macrophage cells. (1488)

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Mol. Cell. Biol. 19, 515-525. Fiber-type-specific transcription of the troponin I slow gene is regulated by multiple elements. 1999

Calvo, S. , Venepally, P. , Cheng, J. , Buonanno, A.

Notes: The authors used the pCAT® Basic and pGL3 Basic Vectors to assay for various slow and fast twitch type troponin I transcriptional regulatory elements. They co-transfected pGL3 Basic-derived Vectors with pRL-SV40 Vector at a ratio of 25:1 using calcium phosphate method. Assayed luciferase activities using Dual-Luciferase® Reporter Assay System in a Berthold luminometer. (1383)

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J. Immunol. 163, 4383-4391. Functional synergism of STAT6 with either NF-κB or PU.1 to mediate IL-4-induced activation of IgE germline gene transcription. 1999

Stutz, A.M., Woisetschlager, M.

Notes: Reporter studies were performed in the human EBV-negative Burkitt's lymphoma cell line DG75. A fragment of the human germline IgE promoter was subcloned into the pGL2-Basic Vector for analysis. The firefly luciferase vector was cotransfected with the pRL-TK Vector at a 20:1 ratio to provide an internal transfection control. The firefly and Renilla luciferases were detected with the Dual-Luciferase® Reporter Assay System. (0307)

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Brain Res. Mol. Brain Res. 69, 62-72. Genomic organization and regulatory elements of the rat latexin gene, which is expressed in a cell type-specific manner in both central and peripheral nervous systems. 1999

Miyasaka, N., Hatanaka, Y., Jin, M., Arimatsu, Y.

Notes: Reporter studies were performed in two modified PC12 cell lines, PC12D and PC12-22a, NS-20 neuroblastoma cells and L6 rat skeletal myoblast-like cells. Promoter constructs were made in the pGL3 Basic Vector. These constructs were cotransfected with the pRL-CMV Vector, a Renilla luciferase expression plasmid, to control for transfection efficiency. The two vectors were transfected at a 100:1 ratio, respectively. The luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. (0696)

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