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J. Gene Med. 5(8), 723-732. Development of a dual-luciferase fusion gene as a sensitive marker for site-directed DNA repair strategies 2003

Bennett, M. and Schaack, J.

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)

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J. Biol. Chem. 278(34), 31895-31901. Down-regulation of hypoxia-inducible factor-2 in PC12 cells by nerve growth factor stimulation. 2003

Naranjo-Suárez, S., Carmen Castellanos, M., Alvarez-Tejado, M., Vara,A., Landázuri, M.O. and del Peso, L.

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)

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Clin. Can. Res. 9, 2933-2939. Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen responsivity of these cells in vivo 2003

Faridi, J., Wang, L., Endermann, G., Roth, R.A.

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® AQueous One Solution Cell Proliferation Assay and showed that the Akt-3 expression confers serum-independent growth on the cells. (2709)

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Clin. Can. Res. 9, 3167-3175. Fully human anti-interleukin 8 antibody inhibits tumor growth in orthotopic bladder cancer xenografts via down-regulation of matrix metalloproteases and nuclear factor-κB 2003

Mian, B.M., Dinney, C.P.N., Bermejo, C.E., Sweeny, P., Tellez, C., Yang, X.D., Gudas, J.M., McConkey, D.J., Bar-Eli, M.

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)

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Plant J. 35(2), 273-283. Gene trapping of the Arabidopsis genome with a firefly luciferase reporter. 2003

Yamamoto, Y.Y., Tsuhara, Y., Gohda, K., Suzuki, K. and Matsui, M.

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)

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J. Biol. Chem. 278, 9332-9338. Identification of an upstream enhancer in the mouse laminin α1 gene defining its high level of expression in parietal endoderm cells. 2003

Niimi, T., Hayashi, Y. and Sekiguchi, K.

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)

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Cancer Res. 63(10), 2658-64. Transactivation of vimentin by beta-catenin in human breast cancer cells. 2003

Gilles C., Polette M., Mestdagt M., Nawrocki-Raby B., Ruggeri P., Birembaut P. and Foidart J.M.

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)

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Am. J. Physiol. Lung Cell. Mol. Physiol. 284(1), L108-18. Transcriptional regulation of CCSP by interferon-gamma in vitro and in vivo. 2003

Ramsay, P.L., Luo, Z., Magdaleno, S.M., Whitbourne, S.K., Cao, X., Park, M.S., Welty, S.E., Yu-Lee, L.Y. and DeMayo, F.J.

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)

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J. Biol. Chem. 278, 5659-5668. Transcriptional Regulation of the Rat NHE3 Gene. Functional Interactions between GATA-5 and Sp family transcription factors. 2003

Kiela, P.R., LeSueur, J., Collins, J.F. and Ghishan, F.K.

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)

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J. Cell Biol. 162, 899-908. Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3–independent-catenin degradation. 2003

Topol, L., Jiang, X., Choi, H., Garrett-Beal, L., Carolan, P.J. and Yang, Y.

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)

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Cancer Res. 62, 5668 – 5671. A novel mechanism of nuclear factor κB activation through the binding between inhibitor of nuclear factor-κBα  and the processed NH2-terminal region of Mig-6. 2002

Tsunoda, T., Inokuchi, J., Baba, I., Okumura, K., Naito, S., Sasazuki, T., and Shirasawa, S.

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)

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Mol. Endocrinol. 16(10), 2243-54. Cloning and characterization of a novel endothelial promoter of the human CYP19 (aromatase P450) gene that is up-regulated in breast cancer tissue. 2002

Sebastian, S., Takayama, K., Shozu, M. and Bulun, S.E.

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)

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Hum. Mol. Genet. 11, 2787-2792. Functional association of the parkin gene promoter with idiopathic Parkinson's disease. 2002

West, A.B., Maraganore, D., Crook, J., Lesnick, T., Lockhart, P.J., Wilkes, K.M., Kapatos, G., Hardy, J.A. and Farrer, M.J.

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)

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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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