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Br. J. Pharmacol. 147, 73–82. Characterization of the Mas-related gene family: structural and functional conservation of human and rhesus MrgX receptors. 2006

Burstein, E.S., Ott, T.R., Feddock, M., Ma, J.N., Fuhs, S., Wong, S., Schiffer, H.H., Brann, M.R. and Nash, N.R.

Notes: This study examined both the human and the macaque counterparts of the Mas-related genes (Mrg), a family of G-protein-coupled receptors, in assays to compare function and structure. Clones of adenylyl cyclase type II (AC2) and Gαo were subcloned into the pSI Mammalian Expression Vector and used in the co-transfection experiments with the Mrgs. Receptor Selection and Amplification Technology (R-SAT™) was used for this comparison of the human and macaque Mrgs. Cells were plated in 96-well plates and transiently transfected with 57ng of 4 different plasmids including one of the Mrg receptors, 2ng AC2 and 30ng pSV-β-galactosidase Vector. One day post-transfection, ligands were added and after 5 days, the β-galactosidase expression was assessed. (3551)

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J. Biol. Chem. 278 (52), 52739-52746. Expression of a human surfactant protein C mutation associated with interstitial lung disease disrupts lung development in transgenic mice.  2005

Bridges, J.P., Wert, S.E., Nogee, L.M. and Weaver, T.E.

Notes: A minimal promoter from the gene encoding BiP was cloned into the pGL3-Basic Vector. The resulting construct was transfected into HEK293 cells and, 48 hours post transfection, cell lysates were prepared using Glo Lysis Buffer. The Bright-Glo™ Luciferase Assay System was then used to assess levels of luciferase expression.  As a transfection control, cells were co-transfected with the pSV-β-Galactosidase Control Vector.  (3229)

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J. Biol. Chem. 279, 29066–29074. BCL-2 translation is mediated via internal ribosome entry during cell stress. 2004

Sherrill, K.W., Byrd, M.P., Van Eden, M.E. and Lloyd, R.E.

Notes: In this paper, the effect of a 5’ untranslated region from the Bcl-2 gene transcripts on firefly and Renilla reporter constructs was evaluated. A number of studies were performed using various single- and dual-reporter constructs containing the Bcl-2 5’ UTR. These constructs were transfected into 293T cells and assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System. Transfection studies with firefly luciferase mRNA constructs were also performed. In these experiments, firefly luciferase levels were measured using the Luciferase Assay System.  Transfections were normalized using the pSV-β-Galactosidase Control Vector and the Beta-Glo® Assay System.  (3125)

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Cancer Res. 64, 7473-7478. Human polynucleotide phosphorylase (hPNPase0ld-35): A potential link between aging and inflammation 2004

Sarkar, D., Lebedeva, I.V., Emdad, L., Kang, D-c., Baldwin, A.S. and Fisher, P.B.

Notes: Human polynucleotide phosphorylase (hPNPaseold-35) was originally identified as a gene that is upregulated during cellular differentiation and senescence. The authors of this study show that overexpression of hPNPaseold-35 results in the increased production reactive oxygen species (ROS) and the activation of the NF-κB pathway, resulting in expression of the inflammatory cytokines IL-6 and IL-8. HeLa cells were transfected with either empty pGL3-Basic or 3kB-Luc (pGL3-Basic containing three tandem NF-κB binding sites). Transfected cells were infected with an empty adenoviral vector or one containing hPNPaseold-35. Luciferase assays were conducted using the Luciferase Assay System, and signal was normalized to a pSV-βgal control. A 10- to 12-fold increase in luciferase activity was observed in the cells infected with the adenovirus containing hPNPaseold-35 compared to the control cells. This activation was inhibited by compounds that reduced ROS. The authors suggest that hPNPaseold-35 plays an important role in producing age-related inflammation. (3643)

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RNA 10, 469-481. Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. 2004

van Eden, M.E., Byrd, M.P., Sherrill, K.W. and LLoyd, R.E.

Notes: The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing. (3429)

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Clin. Can. Res. 9, 5874–5879. Sensitization to the cytotoxicity of cisplatin by transfection with nucleotide excision repair gene Xeroderma Pigmentosun Group A antisense RNA in human lung adenocarcinoma cells. 2003

Wu, X., Fan, W., Xu, S., and Zhou, Y.

Notes: The pGL3-Promoter Vector was treated with various concentrations of the chemotherapeutic cancer and DNA cross-linking agent, cisplatin. The damaged pGL3-Promoter Vector and the pSV-β-Galactosidase Control Vector were then transiently transfected into the human lung adenocarcinoma cell line, A549, and later analyzed for luciferase activity using the Luciferase Assay System with Reporter Lysis Buffer. The researchers also used M-MLV Reverse Transcriptase to clone the antisense sequence to XPA (Xeroderma Pigmentosum group A) mRNA.  The cloned antisense sequence was then expressed in A549 cells for its effect on XPA gene expression. (2833)

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Proc. Natl. Acad. Sci. USA 99, 54–59. Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters. 2002

Ohmichi, T., Maki, A. and Kool, E.T.

Notes: Total RNA was isolated from TOP10F’ or INVaF' E. coli (Invitrogen) using the SV Total RNA Isolation System. The isolated RNA was used in RT-PCR to detect cleavage of marA mRNA. A marA drug resistance gene sequence was also ligated into a chloramphenicol acetyltransferase (CAT) reporter vector. This construct was used to measure single-stranded nanovector-transcribed ribozyme activity on the marA sequence. CAT activity was measured using the CAT Enzyme Assay System. CAT activity was normalized by cotransfecting cells with the pSV-β-Galactosidase Vector and assaying lysates with the β-Galactosidase Enzyme Assay System. (2794)

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J. Biol. Chem. 275, 11666-11671. Distinct roles for recombinant cytosolic 5'-nucleotidase-I and -II in AMP and IMP catabolism in COS-7 and H9c2 rat myoblast cell lines 2000

Sala-Newby, G.B., Freeman, N.V.E., Skladanowski, A.C., Newby, A.C.

Notes: The pTargeT™ Mammalian Expression Vector System was used to clone and express the human cytosolic 5'-nucleotidase II, a 65kDa protein. The protein was expressed and assayed in COS-7 monkey kidney cells and H9c2 rat myoblast cells. The pSV-beta Galactosidase Control Vector was used as a control for transfection efficiency. (0431)

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J. Biol. Chem. 274, 26477–26484.. Functional characterization of the promoter of the x-linked ectodermal dysplasia gene. 1999

Pengue, G., Srivastava, A.K., Kere, J., Schlessinger, D. and Durmowicz, M.C.

Notes: Total RNA was isolated from transfected HeLa cells with the SV Total RNA Isolation System and used for primer extension analysis. Reporter studies were performed in HeLa, 293 and HaCaT cell lines with constructs prepared in the pGL2 Basic and Promoter Vectors. (0006)

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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, 2766-2772. Phytanic acid activates the peroxisome proliferator-activated receptor alpha (PPARAlpha) in sterol carrier protein 2-/sterol carrier protein x-deficient mice. 1999

Ellinghaus, P., Wolfrum, C., Assmann, G., Spener, F., Seedorf, U.

Notes: Reporter studies were performed in rat hepatoma cell line, MH1C1. Experimental constructs were prepared in the pCAT®-3 Basic Vector. Transfections were control by a cotransfection of the pSV-β-Galactosidase Control Vector. (1218)

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J. Biol. Chem. 274, 17789-17793. The mechanism of adenosine formation in cells: Cloning of cytosolic 5'-nucleotidase I 1999

Sala-Newby, G.B., Skladanowski, A.C., Newby, A.C.

Notes: A clone of the cytosolic 5'-nucleotidase I cDNA into the available BamHI-KpnI site of the pTargeT™ Mammalian Expression Vector System. The expression construct was cotransfected into COS-7 cells with the pSV-betaGalactosidase Vector to control for transfection efficiency. (0432)

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J. Immunol. 160, 4896-4903. 3' IgH enhancer elements shift synergistic interactions during B cell development 1998

Ong, J., Stevens, S., Roeder, R.G., Eckhardt, L.A.

Notes: Reporter constructs were prepared in the pGL2 Basic Vector and transfected with a beta-galactosidase vector as a control. Transfections into 18–81, 18–8, M12.4.1, A20, Nama-Iwa and Raji B-cell lines was accomplished with the standard ProFection® Mammalian Transfection System-DEAE Dextran. A lot of detail is provided. The DEAE-Dextran from the kit was also used for a DEAE-Dextran electroporation protocol to transfect for two other B cell lines. A lot of detail is provided. Reporter activities in the transfected cells were determined with the Luciferase Assay System and the Beta-Galactosidase Enzyme Assay System. (0566)

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Mol. Psychiatry 3, 42-49. A family based association study of T102C polymorphism in 5HT2A and schizophrenia plus identification of new polymorphisms in the promoter. 1998

Spurlock, G., Heils, A., Holmans, P., Williams, J., D'Souza, U.M., Cardno, A., Murphy, K.C., Jones, L., Buckland, P.R., McGuffin, P., Lesch, K.P., and Owen, M.J.

Notes: HeLa or SK-N-SH (neuroblastoma) cells (2x105/35 mm) were transfected with reporter constructs using Transfectam® Reagent. Plasmid DNA (5µg) was complexed with 5µl of the reagent and left on the cells for 24 hours. Promoters were induced with PMA and activity was measured using a luciferase assay. The transfection control was the pSV-Beta-Galactosidase Control Vector. (0365)

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Oncogene 16, 423-428. C-Myc 5' untranslated region contains an internal ribosome entry segment. 1998

Stoneley, M., Paulin, F. E., Le Quesne, J. P., Chappell, S. A., Willis, A. E.

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)

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Am. J. Hum. Genet. 63, 717-726. Dihydropyrimidinase deficiency: structural organization, chromosomal localization, and mutation analysis of the human dihydropyrimidinase gene. 1998

Hamajima, N., Kouwaki, M., Vreken, P., Matsuda, K., Sumi, S., Imaeda, M., Ohba, S., Kidouchi, K., Nonaka, M., Sasaki, M., Tamaki, N., Endo, Y., De Abreu, R., Rotteveel, J., van Kuilenburg, A., van Gennip, A., Togari, H., Wada, Y.

Notes: pSV-β-Galactosidase Control Vector was cotransfected as an internal standard. Cell extracts were made 24 hours after the transfection using the Reporter Lysis Buffer and assayed using the β-Galactosidase Enzyme Assay System . (1056)

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Hepatology 28, 1147-1153. Mutations in the interferon-sensitivity determining region of hepatitis C virus and transcriptional activity of the nonstructuraql region 5A protein. 1998

Fukuma, T., Enomoto, N., Marumo, F., Sato, C.

Notes: The ProFection® Mammalian Transfection System-Calcium Phosphate was used to transfect reporter plasmids into HuH-7, a human hepatoma cell line. The cells were plated at 1.2 X 106 in a 100mm dish and transfected 48 hours later. Transfection efficiency was monitored with a co-transfection of the pSV-β-Galactosidase Control Vector with the CAT reporter vectors. (1130)

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J. Biol. Chem. 273, 32312-32321. Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transciptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA 1998

Newton, R., Seybold, J., Kuitert, L.M.E., Bergmann, M. , Barnes, P.J.

Notes: A reporter construct was assembled in the pGL3 Control Vector downstream of the Xba I consisting of the 3'UTR of the COX-2 gene. The construct was designed to see if the UTR had an affect on mRNA stability in the presence of IL-1beta. The construct as well as the pSV-Beta Galactosidase Control Vector were transfected into A549 cells using the Tfx™-50 Reagent. A lot of detail is provided for the transfection. The reporter activities were monitored with the Luciferase Assay System and the Beta-Galactosidase Enzyme Assay System. (0606)

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J. Biol. Chem. 273, 26218-26224. Synergistic activation of the N-methyl-D-aspartate receptor subunit 1 promoter by myocyte enhancer factor 2C and Sp1. 1998

Krainc, D., Bai, G., Okamoto, S., Carles, M., Kusiak, J.W., Brent, R.N., Lipton, S.A.

Notes: Luciferase (prepared in pGL2-Basic Vector) and β-galactosidase (pSV-β-Galactosidase Control Vector) reporters were studied in SL2 Drosophila cells, HeLa cells and primary rat cortical neurons. Transfections into the SL2 and HeLa cells were accomplished with standard calcium phosphate methods. Transfection of primary rat cortical neurons was accomplished with the Tfx™-50 Reagent as described in Boeckman, F.A. et al. (1996) Neural Notes II(1), 13-15. Reporter assays were performed with the Luciferase Assay System and the β-Galactosidase Assay System. (0890)

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Hepatology 28, 1013-1021. The hepatitis B virus X protein up-regulates tumor necrosis factor alpha gene expression in hepatocytes. 1998

Lara-Pezzi, E., Majano, P.L., Gómez-Gonzalo, M., García-Monzón, C., Moreno-Otero, R., Levrero, M., López-Cabrera, M.

Notes: Transfections of HepG2 cells, 2.2.15 (a Hepatitis B-infected cell line) and HeLa cells were accomplished with the ProFection® Mammalian Transfection System-Calcium Phosphate. Transfection efficiencies were monitored with co-transfected pSV-β-Galactosidase Control Vector, but the efficiencies were not reported. (0843)

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J. Biol. Chem. 272, 22667-22678. A minimal murine Msx-1 gene promoter. organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice. 1997

Takahashi, T., Guron, C., Shetty, S., Matsui, H., Raghow, R.

Notes: Expression of firefly luciferase gene fused with the promoter of interest was performed in NIH3T3 and C2C12 myoblasts. The Altered Sites® II in vitro Mutagenesis System was used to produce site-directed mutations within the promoter. (0294)

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J. Biol. Chem. 272, 21045-21051. A type I interferon signaling factor, ISF21, encoded on chromosome 21 is distinct from receptor components and their down-regulation and is necessary for transcriptional activation of interferon-regulated genes. 1997

Holland, K.A., Owczarek, C.M., Hwang, S.Y., Tymms, M.J., Constantinescu, S.N., Pfeffer, L.M., Kola, I., Hertzog, P.J.

Notes: Luciferase and CAT assays were conducted in CHO-K1 cells using constructs prepared in the pGL3-Basic and pCAT®-Basic Vectors, and the activity was normalized to β-galactosidase activity supplied by the pSV-β-Galactosidase Control Vector. The pCAT® Vectors have been replaced by the next-generation pCAT®3 Vectors. (1055)

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J. Biol. Chem. 272, 15993-16001. Activation transcription factor 1 involvement in the regulation of murine H-2Dd expression. 1997

Ishiguro, N., Brown, G.D., Meruelo, D.

Notes: pSV-β-Galactosidase Control Vector was used as a transfection control in F9 cells and TNT® T7 Coupled Reticulocyte Lysate System was used to translate ATF-1 protein for use in gel shift assays. (1004)

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J. Biol. Chem. 272, 31793-31800. CCAAT/Enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification Of a novel cyclic amp signaling pathway in bone 1997

Umayahara, Y., Ji, C., Centrella, M., Rotwein, P., McCarthy, T.L.

Notes: Studies were performed in COS-7 cells. The experimental firefly luciferase vector (derived from pGL2-Basic Vector) was co-transfected with the Renilla control Vector (pRL-CMV Vector) and plasmids expressing either the CCAAT/Enhancer-binding protein beta or delta from a CMV promoter. The firefly luciferase:expression plasmid ratio was 10:1 and the firefly luciferase:Renilla luciferase vector ratio was 1000:1. In vitro translations were performed with the TNT® Coupled Reticulocyte Lysate System. (0252)

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J. Biol. Chem. 272, 16498-16506. Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products 1997

Li, J., Schmidt, A.M.

Notes: pGL3 Basic Vector and pSV-β-galactosidase Control Vector were used in bovine aortic endothelial cells and rat vascular smooth muscle cells. The NF-κB p50 protein was used for DNase Footprinting (2-20 gel shift units). HeLaScribe® Nuclear Extract, Gel Shift Grade, was used as a positive control for NF-κB in a gel shift assay. (0793)

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