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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, 42280–42288. GAGA factor down-regulates its own promoter. 2002

Kosoy, A., Pagans, S., Espina´s, M.L., Azorýn, F. and Bernue´s, J.

Notes: Researchers PCR amplified and cloned the Trl promoter into the pGEM®-T vector. The Trl promoter sequence, as well as the eve promoter sequence and/or a CMV promoter, were used to make luciferase reporter constructs using the pGL3-Basic Vector. Transient transfection experiments were performed with the promoter constructs and GAGA factor expression constructs. Relative luciferase activity from each experiment was measured and compared to that of a control.  (2775)

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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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Cancer Res. 62(15), 4316-4324. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. 2002

Rapisarda, A., Uranchimeg, B., Scudiero, D.A., Selby, M., Sausville, E.A., Shoemaker, R.H. and Melillo, G.

Notes: The authors of the study undertook a screen of small molecules to find chemicals that reduce gene expression caused by hypoxia. Neomycin resistant stable cell lines derived from U251 human glioma cells expressing luciferase under the control of either the HSV thymidine kinase (TK) with or without hypoxia-response element (HRE) repeats, or the SV40 promoter were constructed.  U251 cell lines expressing luciferase under control of either a vascular endothelial growth factor (VEGF) promoter or a minimal HRE containing VEGF promoter were also tested.  To screen for inhibitors of the hypoxia induced pathway, 3,000 cells in 25µl/well were plated in 384-well plates using a Beckman Coulter Biomek® 2000 automated workstation. Cells were cultured for 24 hours under normal conditions, then compounds from the NCI "Diversity Set" of approximately 2,000 compounds were added (in 25µl of carrier), and the cells were placed in a hypoxia chamber for 20 hours. Cells were allowed to recover for 1.5 hours in normoxic conditions before addition of 40µl/well  Bright-Glo™ Luciferase Assay Reagent.  Luminescence was recorded using a Tecan Ultra Multifunction Plate Reader in luminescence mode.  Further analyses of compound "hits" were performed on the VEGF-promoted luciferase cell lines in 96-well plates. (3096)

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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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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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J. Biol. Chem. 276(45), 41690-9. Transcriptional regulation of the human sterol 12alpha-hydroxylase gene (CYP8B1): roles of heaptocyte nuclear factor 4alpha in mediating bile acid repression. 2001

Zhang, M. and Chiang, J.Y.

Notes: To study the transcriptional control of human CYP8B1 by bile acids, the -514/+300 region of the gene was cloned into the pGL3-Basic Vector and subsequently mutagenized at both the 5´ and 3´ends of the CYP8B1 fragment.  The various reporter constructs were then transfected into HepG2 cells and the activity assessed using the Luciferase Assay System. Luminescence was determined using a Berthold Lumat LB 9501 luminometer. The Gal4/Id and VP16/MyoD provided in the CheckMate™ Mammalian Two-Hybrid System were used as positive controls for the in a two-hybrid assay that examined the interaction of SHP and HNF4alpha, genes that regulate CYP8B1.  The TNT® Quick Coupled Transcription/Translation System was used to synthesize various receptor proteins cloned in expression plasmids. (3083)

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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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Eur. J. Biochem. 267, 7224–7229. Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifolii 2000

Lee, H.Y., An, J.H. and Kim, Y.S.

Notes: Total RNA was isolated from Rhizobium leguminosarum bv. trifolii using the SV Total RNA Isolation System. This RNA was used in a primer extension assay using AMV Reverse Transcriptase to determine the transcriptional start site of the mat operon. Nested deletions during the sequencing of the mat promoter were prepared. The firefly luciferase gene from pGL3 Basic was used to construct a reporter construct containing the mat promoter to study transcriptional effects of MatR. The interaction of the MatR protein with the mat operator was mapped. (2308)

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J. Biol. Chem. 275, 24847-24856. Inhibitory and stimulatory effects of lactacystin on expression of nitric oxide synthase type 2 in brain glial cells. 2000

Stasiolek, M., Gavrilyuk, V., Sharp, A., Hovarth, P., Selmaj, K. and Feinstein, D.L.

Notes: In this study, a fragment of the nitric oxide synthase type 2 (NOS2) promoter was cloned into the pGL3-Basic Vector. C6 glioma cells stably transfected with this construct were then exposed to various NOS2 inducers and lactacystin. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System. (2435)

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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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Am. J. Respir. Cell Mol. Biol. 22(5), 582-589. Molecular regulation of granulocyte macrophage colony-stimulating factor in human lung epithelial cells by interleukin (IL)-1beta, IL-4, and IL-13 involves both transcriptional and post-transcriptional mechanisms. 2000

Bergmann, M., Barnes, P.J. and Newton, R.

Notes: The interleukin (IL)-1ß stimulated release of granulocyte macrophage colony stimulating factor (GM-CSF) from lung epithelial cells was explored in this study. To test the promoter activity of GM-CSF, the promoter and enhancer regions were amplified by PCR from human peripheral blood mononuclear cells and cloned into the pGEM®-T vector. After verification by sequencing, the promoter and enhancer were cloned individually and together into the pGL3-Basic Vector. Additionally, an Xho I/Sal I fragment containing the HSV tk promoter, a gene conferring neomycin resistance, and a poly-A tail were cloned into the Sal I site of pGL3 to allow the production of stable transfectants. To perform stable transfections, 20µl of the Tfx™-50 transfection reagent was incubated with 8µg of plasmid in serum-free medium for 15 minutes at room temperature.  Preconfluent human A549 type II alveolar carcinoma cells were incubated with the transfection mix for 2 hours after washing with serum-free medium. The cells were then cultured in fresh medium for 16 hours before the addition of  0.5mg/mL G-418. After 21 days, foci of cells developed and were harvested for use in luciferase assays. The cells were plated into 24-well plates, grown to confluency and incubated in serum-free medium. Stimulation by 1ng/ml IL-1ß or 1µM PMA for 12 hours was followed by lysate production and measurement of luciferase activity using the Luciferase Assay System and a Turner luminometer. Luciferase readings were standardized against total protein measurements. The PolyATract® System IV was also used prior to a Northern Blot to obtain purified mRNA. (2742)

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EMBO J. 18, 6973-6982. Cell type-specific activation of mitogen-activated protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting cells. 1999

Hacker, H., Mischak, H., Hacker, G., Eser, S., Prenzel, N., Ullrich, A., Wagner, H.

Notes: Treatment of RAW 264.7 cells were treated with CpG-oligonucleotides derived from bacterial DNA. The cells responded by releasing TNFα and IL-12. The MEK Inhibitor U0126 inhibited the release of the proteins. The inhibitor has no effect on the TNFα promoter or mRNA levels but has a big effect on IL-12 promoter activity and mRNA levels, suggesting different mechanisms of MAPK pathway involvement. Promoter studies were performed with constructs made in the pGL3-Basic Vector and analyzed with the Luciferase Assay System. (1091)

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J. Biol. Chem. 274, 11220-11228. Cloning of the cyclin A1 genomic structure and characterization of the promoter region. GC boxes are essential for cell cycle-regulated transcription of the cyclin A1 gene. 1999

Muller, C., Yang, R., Beck-von-Peccoz, L., Idos, G., Verbeek, W. and Koeffler, H.P.

Notes: The promoter of the human cyclin A1 gene was studied by the authors of this work. To define promoter activity, various genomic fragments from upstream of the cyclin A1 gene were analyzed in the pGL3-Basic luciferase vector. These constructs were expressed in HeLa cells and in the Drosophila cell line S2. For studies in S2 cells, an expression vector encoding the transcription factor Sp1 was also used. The data for HeLa cells is displayed as fold activation over that of the pGL3-Basic Vector without insert normalized against a CMV driven β-galactosidase control. No β-galactosidase control was used in S2 cells because the viral promoter control vectors all cross-reacted with the Sp1 expression vector. The data in S2 cells is expressed as fold activation over similar transfections without Sp1. (3002)

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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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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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J. Biol. Chem. 274, 19699-19706. Identification and characterization of a ran gene promoter in the protozoan pathogen Giardia lamblia 1999

Sun, C.-H., Tai, J.-H.

Notes: The pSP-luc+ Vector was used as a source of luciferase cDNA for construction of a expression vector in G. lamblia. The promoter of interest was combined with luc+ in a standard cloning vector for study. Deletion mutants of the promoter were constructed with the Erase-a-Base® System. The authors note that the pGL3 Control Vector produced some luciferase activity in the G. lamblia but only 0.5% of the RAN promoter-luc+ construct. (0317)

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J. Biol. Chem. 274, 3970-3977. Identification of a functional peroxisome proliferator-responsive element in the murine fatty acid transport protein gene. 1999

Frohnert, B. I. , Hui, T. Y. , Bernlohr, D. A.

Notes: The promoter region (and 5' deletion variants thereof) was inserted upstream of the firefly luciferase gene in pGL3 Basic Vector. These constructs were co-transfected into CV-1 cells with various different PPAR isoform expression plasmids and pRL-SV40 Vector as a transfection control at a ratio of 50: 25: 1, respectively. Luciferase activities were determined using the Dual-Luciferase® Reporter Assay System . (1125)

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J. Immunol. 162, 5337-5344. NF-κB-inducing kinase is a common mediatorof IL-17, TNF-α, and IL-1-β- induced chemokine promoter activation in intestinal epithelial cells 1999

Awane, M., Andres, P.G., Li, D.J., Reinecker, H-C.

Notes: IEC-6 cells were seeded into 96-well plates and cultured in the presence of various concentrations of human IL-17. Cell proliferation was measured using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. The pGL3 vector was used to clone a PCR-amplified piece of the 5' flanking region of the CINC gene, and the Luciferase Assay System was used to assess promoter activation. All transfections of IEC-6 cells used the pSV-β-galactosidase vector as a control for transfection efficiency. Total RNA for Northern blotting was isolated using the Poly ATtract System. (2510)

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J. Biol. Chem. 274, 1277-1285. Promoter characterization of the rat gene for Ca2+-binding protein regucalcin. Transcriptional regulation by signaling factors. 1999

Murata, T., Yamaguchi, M.

Notes: Reporter studies were performed in rat H4-II-E hepatoma cells. Various 5' deletion series of rat regucalcin gene promoter were prepared in the pGL3 Basic Vector. The pGL3-based constructs were cotransfected with pRL-TK Vector at a 4:1 ratio and both firefly and Renilla luciferase activities determined with the Dual-Luciferase® Reporter Assay System. All transfections were performed with the Tfx™-20 Reagent. (0632)

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Biochemistry 37, 6533-6540. A novel tetraester construct that reduces cationic lipid-associated cytotoxicity, implication for the onset of cytotoxicity 1998

Aberle, A.M., Tablin, F., Zhu, J., Walker, N.J., Gruenert, D.C. and Nantz, M.H.

Notes: Various lipid formulations were used to transfect NIH 3T3 cells with the pGL3 Control Vector to monitor the transfection. The cytotoxic effect of these lipid combinations were measured with the CellTiter 96® AQueous Proliferation Assay (MTS/PMS). (2054)

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