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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. 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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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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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, 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, 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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Proc. Natl. Acad. Sci. USA 94, 9487-9492. Characterization of the α1B-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle. 1997

Eckhart, A.D. , Yang, N. , Xin, X. , Faber, J.E.

Notes: Luciferase studies were performed in aortic and vena caval smooth muscle cells. Transfections were normalized to β-galactosidase activity. Researchers report that pGL3 Promoter and pGL3 Control Vectors were 55-± 3-fold and 340±41-fold greater activity, respectively, than the pGL3 Basic Vector alone. The CREB consensus oligo was used in gel shifts of aortic and vena cava smooth muscle cell nuclear extracts. (1214)

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J. Biol. Chem. 272, 12692-12698.. Cloning and characterization of the promoter region of a gene encoding a 67-kDa glycoprotein. 1997

Chatterjee, N. , Zou, C. , Osterman, J. C. , Gupta, N. K.

Notes: Luciferase studies were performed in KRC-7 rat hepatoma cells using constructs prepared in the pGL3 Basic Vector. The luciferase constructs were cotransfected with the pSV-beta-galactosidase Vector. Luciferase activities determined with the Luciferase Assay System were normalized to readings from the Beta-Galactosidase Enzyme Assay System. (1364)

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J. Biol. Chem. 272, 22425-22431. Cloning and characterization of the Type I inositol 1,4,5-trisphosphate receptor gene promoter. regulation by 17β-estradiol in osteoblasts. 1997

Kirkwood, K.L., Homick, K., Dragon, M.B., Bradford, P.G.

Notes: CAT reporter studies were performed in G-292 osteosarcoma cells using constructs prepared in the pCAT®-Basic Vector. CAT activity was normalized to β-Galactosidase activity. (0903)

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J. Biol. Chem. 272, 5915-5920. Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes. 1997

Price, D. J., Rivnay, B., Fu, Y., Jiang, S., Avraham, S., Avraham, H.

Notes: The pSV-β-Galactosidase Vector was used as transfection control. The protein of interest was detected by western blotting. The amount of protein loaded on the gel was normalized to β-galactosidase activity. (0547)

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Mol. Pharmacol. 51, 703-710.. Effect of transforming growth factor-beta1 on expression of aryl hydrocarbon receptor and genes of Ah gene battery: clues for independent down-regulation in A549 cells. 1997

Dohr, O. , Sinning, R. , Vogel, C. , Munzel, P. , Abel, J.

Notes: The Transfectam® Reagent was used to transiently transfect A549 human lung cancer cells. Many details of the transfection are provided. Luciferase activity was normalized to Beta Galactosidase activity. (1240)

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J. Biol. Chem. 272, 29821-29828. Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. 1997

Ikonen, T., Palvimo, J.J., Janne, O.A.

Notes: The authors used the Promega pSV-β-Galactosidase Control Vector, pGL3-Control and pGL3-Basic Vectors, Luciferase Assay Reagent, TNT® Coupled Wheat Germ Extract Systems and TNT® Coupled Reticulocyte Lysate Systems in their studies NF-κB. (0994)

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J. Biol. Chem. 272, 15003-15010. Isolation and characterization of the human gp130 promoter. Regulation by STATS. 1997

O'Brien, C. A. , Manolagas, S. C.

Notes: Luciferase studies performed in HepG2 cells using constructs prepared in the pGL3 Basic Vector. The luciferase constructs were cotransfected with the pSV-beta-Galactosidase Control Vector to normalize for transfection efficiency. Luciferase activities were measured with the Luciferase Assay System. (0629)

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