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Clin. Can. Res. 10, 7547-7554. Epidermal growth factor receptor tyrosine kinase inhibition represses cyclin D1 in aerodigestive tract cancers.  2004

Petty, W.J., Dragnev, K.H., Memoli, V.A., Ma, Y., Desai, N.B., Biddle, A., Davis, T.H., Nugent, W.C., Memoli, N., Hamilton, M., Iwata, K.K., Rigas, J.R. and Dmitrovsky, E.

Notes: The CellTiter-Glo® Luminescent Cell Viability Assay was used to examine the proliferation of BEAS-2B (immortalized human bronchial epithelial (HBE)) cells after treatment with various concentrations of erlotinib, an inhibitor of the mitogenic effects of EGF. For these experiments, 3,000/well were seeded in 96-well plates and incubated for 72 hours with various concentrations of erlotinib. Other lung cell carcinoma cell lines (A549, H226, H358, and H441) were also tested for proliferation in the presence of erlotinib using the CellTiter-Glo® Assay system. The researchers used a luciferase construct that contained the cyclin D1 promoter to test the effects of erlotinib on cyclin d1 regulation. This construct, along with the pRL-TK vector, were co-transfected into BEAS-2B cells. Groups of cells were then treated with or without EGF and erlotinib and assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System.  (3258)

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J. Biol. Chem. 279(21), 22228-35. Identification of a novel PDX-1 binding site in the human insulin gene enhancer. 2004

Le Lay, J., Matsuoka, T.A., Henderson, E. and Stein, R.

Notes: The GG2 element located upstream of the human insulin gene was mutated and cloned into a firefly luciferase construct. Two pancreatic mouse cell lines, ßTC-3 and Min6, were co-transfected with the various GG2 luciferase vectors using phRL-TK Vector as a normalization control. Luciferase expression was then assessed using the Dual-Luciferase® Reporter Assay System. Three factors known to affect insulin gene expression were transcribed and translated using the TNT® Coupled Reticulocyte Lysate System. The proteins were then used in a gel-shift assay with several DNA element oligos. A 38-40 kDa protein that bound to the GG2 element was identified.  This protein was isolated by DNA affinity chromatography, run on a SDS-PAGE gel and digested with 0.01µg/µl Sequencing Grade Modified Trypsin. Digestion products were then analyzed by Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and TOF/TOF tandem mass spectrometry. (3116)

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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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RNA 10, 277–286. Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene. 2004

Verge´, V., VonLanthenm, M., Masson, J.M., Trachsel, H., and Altmann, M.

Notes: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM®-T Vector. (2845)

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J. Biol. Chem. 279(18), 19091-8. Myeloid Elf-1-like factor, an ETS transcription factor, up-regulates lysozyme transcription in epithelial cells through interaction with promyelocytic leukemia protein. 2004

Suico M.A., Yoshida H., Seki Y., Uchikawa T., Lu Z., Shuto T., Matsuzaki K., Nakao M., Li J.D. and Kai H.

Notes: These authors investigated the role of the promyelocytic leukemia (PML) nuclear body in transactivation of myeloid elf-1-like factor (MEF), a transcription factor that upregulates lysozyme transcription. To determine if the nuclear factors affected MEF, HeLa cells were cotransfected with 0.2µg of a pGL2 Vector construct with a lysozyme promoter and various combinations of 0.1µg of MEF, 0.5µg of PML and 1µg of Sp100 (another nuclear body factor) plasmids. Expression was normalized to 10ng of phRG-TK Vector. Forty-eight hours post-transfection, the cells were harvested and luciferase activity measured using the Dual-Luciferase® Reporter Assay System in a Turner Designs luminometer model TD-20/20. In addition, various MEF mutants were made and tested in the same dual-reporter system to determine if transactivation was affected by the various deletion mutations created. The same MEF mutants were also cloned into a vector with the yeast GAL4 DNA-binding domain to help determine what domain of MEF was interacting with PML in a mammalian two-hybrid system. This was done by creating pACT-PML and using the CheckMate™ Mammalian Two-Hybrid System. (3086)

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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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Mol. Cell. Biol. 24(8), 3337-46. Recruitment of N-CoR/SMRT-TBLR1 corepressor complex by unliganded thyroid hormone receptor for gene repression during frog development. 2004

Tomita A., Buchholz D.R. and Shi Y.B.

Notes: The authors used Xenopus laevis oocytes to show that unliganded thyroid hormone receptor (TR) recruits N-CoR (nuclear receptor corepressor) to modulate metamorphosis.  To study this influence, the cytoplasm of stage VI oocytes from X. laevis was injected with the indicated mRNAs [TR, retinoic acid receptor (RXR) and FLAG-tagged N-CoR].  The reporter plasmid TRE-Luc (0.33 ng/oocyte; thyroid hormone response elements from a Xenopus promoter driving expression of the firefly luciferase gene) and the control vector phRG-TK (0.03 ng/oocyte) were co-injected into the germinal vesicle (nucleus) after mRNA injection. After overnight incubation at 18°C, oocyte lysates were prepared by lysing six oocytes in 90µL 1X Passive Lysis Buffer. The Dual-Luciferase® Reporter Assay System was then used to assay 7µl of the lysate. The researchers also used an expression vector (based on the pGEM®-4Z Vector) containing the 5’ and 3’ untranslated regions of the X. laevis beta-globin gene flanking the multiple cloning site. (3088)

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Hum. Mol. Genet. 13, 2221-2231. SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease. 2004

Shirasawa, S., Harada, H., Furugaki, K., Akamizu, T., Ishikawa, N., Ito, K., Ito, K., Tamai, H., Kuma, K., Kubota, S., Hiratani, H., Tsuchiya, T., Baba, I., Ishikawa, M., Tanaka, M., Sakai, K., Aoki, M., Yamamoto, K. and Sasazuki, T.

Notes: Real-time TaqMan® amplification reactions were cleaned up using the Wizard® MagneSil® Sequencing Reaction Clean-Up System.  The purified products were then used in sequencing reactions.  This paper also describes use of the Dual-Luciferase® Reporter Assay System to analyze HEK293 cells transfected with a pGL3-Enhancer vector construct.  (3181)

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Curr. Biol. 12, 2162-2167. The Human DiGeorge Syndrome Critical Region Gene 8 and Its D. melanogaster Homolog Are Required for miRNA Biogenesis 2004

Landthaler, M., Yalcin, A., and Tuschl, T.

Notes: In this study, the psiCHECK-2 vector was used to assist in selection of siRNA sequences that work optimally against their selected target. Dicer and DGCR8 coding sequences were individually cloned into the psiCHECK-2 multiple cloning site. The resulting vector and a synthetic siRNA duplex targeting the gene coding sequence were transfected into 293 cells and  Renilla luciferase activity was measured using the Dual Luciferase® Assay System. Firefly luciferase activity was used to normalize the data.  siRNAs that caused greater than 80% reduction in Renilla luciferase signal were selected for further use. (3220)

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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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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.


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J. Biol. Chem. 278, 7445–7452. Microtubule disruption utilizes an NFkappa B-dependent pathway to stabilize HIF-1alpha protein. 2003

Jung, Y.J., Isaacs, J.S., Lee, S., Trepel, J. and Neckers, L.

Notes: In this paper, the role of microtubules on regulation of hypoxia-inducible factor (HIF) 1-α was explored. A549 human lung cancer cells were transiently transfected with 5µg of NFκB super-repressor plasmid or 3µg of either HA-tagged wild type or mutated HIF-1α construct using FuGENE® 6 Transfection Reagent in 6cm dishes. Twenty-four hours post-transfection, the cells were treated with vinblastine or colchicine before lysing the cells and analyzing the lysate by Western blotting using anti-HIF-1α or anti-HA antibodies (Figures 3 and 6 in article). A549, hepa1c1c7 and hepa1c4 cells were transiently cotransfected with 0.4µg of an iNOS luciferase construct or an NFκB-dependent luciferase vector and 4ng of a CMV Renilla luciferase plasmid in six-well plates. After six hours, the cells were treated with vinblastine, colchicine, nocodazole and paclitaxel and incubated for an additional 6–10 hours. The luciferase activity was then assessed using the Dual-Luciferase® Reporter Assay System (Figures 2 and 4). (4293)

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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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