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J. Biol. Chem. 279, 49617-49623. Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3 pathway. 2004

Yamaguchi, K., Lee, S.H., Eling, T.E., and Baek, S.J.

Notes: The LY294002 phosphatidylinositol 3-kinase (PI3K) inhibitor was used to identify Nonsteroidal Anti-inflammatory Drug-activated Gene (NAG-1) as a novel downstream target of the PI3K pathway during cell activation. For these experiments, HCT-116 cells were treated with 50μM LY294002 and NAG-1 protein expression was assessed by Western blotting. Gene upregulation during LY294002 treatment was measured with a luciferase reporter construct containing the NAG-1 promoter, the pRL-null Vector as a transfection control, and the Dual-Luciferase® Reporter Assay System. (3262)

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Plant Cell 16(5), 1235-1250. Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. 2004

Dunoyer P., Lecellier C.H., Parizotto E.A., Himber C. and Voinnet O.

Notes: The authors cloned five distinct silencing suppressor proteins from five different plant viruses in order to examine the pathways involving both small interfering RNA and micro RNA in Arabidopsis thaliana. These viral factors [P1- HcPro of Turnip mosaic virus (TuMV), P38 protein of Turnip crinkle virus (TCV), P19 protein of Tomato bushy stunt virus(TBSV), P25 protein of Potato virus X, and the P15 protein of Peanut clump virus (PCV)] were inserted into a mammalian expression vector and tested for protein production using the TNT® Quick Coupled Transcription/Translation System. The suppression effects of these plant viral proteins were also tested in HeLa cells. The CMV promoter was cloned from pRL-CMV into the pGL3-Basic Vector and both plasmids were transfected at 500ng each plus 1µg of each of the five suppressor-expressing vectors. After one day, 300ng siRNA targeting the firefly luciferase gene was added. Twenty-four hours later, the Dual Luciferase® Reporter Assay System was used to determine the ratio of firefly:Renilla luciferase expression and see if the viral suppressor protein had an effect. (3085)

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Endocrinology 144, 4410–9. Activin βC-subunit heterodimers provide a new mechanism of regulating activin levels in the prostate. 2003

Mellor, S.L. et al.

Notes: The authors investigated the ability of activin proteins to regulate a reporter gene under the control of androgen response elements by transiently transfecting 50,000 CHO cells per well in 24-well plates at 70–80% confluence for 24 hours using the FuGENE® 6 Transfection Reagent at a ratio of 1:3 (µg of reporter plasmid to µg of reagent). The authors also examined the effect of activins on a reporter gene under the control of GnRH receptor-activating sequences by transiently transfecting 500,000 LβT2 cells per well in 24-well plates at 80–80% confluence for 24 hours using the FuGENE® 6 Transfection Reagent at a DNA:reagent ratio of 1:3. In both cases, reporter gene activity was measured 24 hours post-transfection. (4387)

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pRL-CMV Vector

Mol. Cancer Res. 1, 475-84. Gene expression profiling in prostate cancer cells with Akt activation reveals Fra-1 as an Akt-inducible gene. 2003

Tiwari, G., Sakaue, H., Pollack, J.R., and Roth, R.A.

Notes: These authors analyzed gene expression profiles in the prostate cancer cell line PC3 upon induction of Akt activity to try to identify genes regulated by Akt that participate in the transformation of cells. They identified one mRNA of interest (Fra-1) and cloned its 5' regulatory region into a pGL3-Basic firefly luciferase reporter construct. This construct was used to transiently transfect MCF7 human breast cancer cells along with an Akt plasmid construct and a control vector expressing Renilla luciferase. The firefly construct was induced 4- to 5-fold by co-transfection with Akt3. Transfection conditions were as follows: MCF-7 cells were grown to 70% confluence in six-well plates, then incubated for 15 min with a mixture of 5ng of the control Renilla plasmid, 0.5μg of the Akt-expressing plasmid, and 0.5μg of pFra-luc construct and Fugene® 6 reagent at a 3:1 transfection reagent:DNA ratio. After 48 hours, luciferase activity was assessed using the Dual-Luciferase® Reporter Assay System.


(4361)

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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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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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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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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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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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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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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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Proc. Natl. Acad. Sci. USA 99, 1443-1448. Stable suppression of gene expression by RNAi in mammalian cells. 2002

Paddison, P. J., Caudy, A. A., and Hannon, G. J.

Notes: Firefly and Renilla luciferase mRNA transcripts were synthesized with the Riboprobe® kit for use in in vitro translation and dicer experiments.  The Dual-Luciferase® Assay was used to analyze cotransfection experiments with the pRL-SV40 and pGL3-Control Vectors, and dsRNAs in P19 mouse embryonyl carcinoma cells. (2621)

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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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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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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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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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Proc. Natl. Acad. Sci. USA 96, 151-156. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. 1999

Xu, Y. , Piston, D. W. , Johnson, C. H.

Notes: Promega's pRL-null Vector was used as a source of the Renilla luciferase gene to make C-terminal fusions to this gene. This was used in concert with C-terminal fusions made to the EYFP gene (CLONTECH). Protein:protein interactions between the fusion partners of Renilla luciferase and EYFP brought these proteins close enough together to allow energy transfer of the bioluminescent signal generated by Renilla luciferase to excite EYFP, which then emitted light at its wavelength (527 nm). (0128)

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Products

pRL-null Vector

Cell 97(1), 29-39. A Smad transcriptional corepressor. 1999

Wotton, D., Lo, R.S., Lee, S. and Massague, J.

Notes: The pRL-CMV plasmid was used as an internal transfection efficiency control. The authors used a homebrew Renilla assay system. The buffer used was 25mM Tris (pH 7.5), 100mM NaCl, with 0.09µM coelenterazine (Biosynth) from an 0.09µM stock in 20mM HCl in methanol. (2170)

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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, 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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