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Cell 121, 1097–108. Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. 2005

Leaman, D., Chen, P.Y., Fak, J., Yalcin, A., Pearce, M., Unnerstall, U., Marks, D.S., Sander, C., Tuschl, T. and Gaul, U.

Notes: To monitor the effects of targeted degradation of micro RNA (miRNA) on Drosophila embryos, the authors cloned the full-length 3'UTRs of the proapoptotic factors hid and reaper into the psiCHECK™-2 Vector. The constructs were injected as plasmids (1µg/ml) mixed with 400µM sense and antisense miR-2 2'O-methyl oligoribonucleotides in early embryos. The total volume injected was equal to 5% of egg volume. After 10 hours development, the embryos were washed and lysed under agitation using 60µl lysis buffer and shaken at 750 rpm at room temperature for 30 minutes. The resulting lysate was cleared by centrifugation and three aliquots were tested using the Dual-Luciferase® Reporter Assay System. The Renilla-to-firefly luciferase ratios from three to five independent replicates were averaged and normalized to the value of the miR-6 sense control, the most severe apoptotic phenotype when targeted for depletion. Statistical significance was assessed using the t test. (3292)

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Proc. Natl. Acad. Sci. USA 102, 12759-12764. Biological function of the vaccinia virus Z-DNA-binding protein E3L: gene transactivation and antiapoptotic activity in HeLa cells. 2005

Kwon, J.A. and Rich, A.

Notes: The host gene E3L is required for vaccinia virus infection and has anti-apoptosis activity. The authors examine the ability of E3L to regulate transcription of several genes related to apoptosis, immune response and viral pathogenesis. IL-6, NF-AT, p53, NF-κB, Ap-1 and cAMP response elements were cloned upstream of a TATA box and the firefly luciferase reporter gene. Renilla luciferase (pRL-null Vector) was used to normalize for transfection efficiency. Luciferase activities were measured using the Dual Luciferase® Reporter Assay System. The authors also show that the Z-DNA binding region of E3L is important for transcriptional regulation. HeLa cells were transfected with an expression vectors expressing full-length E3L, E3L with a deletion of the Z-DNA binding domain or E3L with point mutations in residues important for Z-DNA binding. The AccessQuick™ RT-PCR System was used to quantitate IL-6, NF-AT and p53 mRNAs; β-actin was used as a control for RNA input. (3452)

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J. Biol. Chem. 280, 28215-28220. Determination of the functionality of common APOA5 polymorphisms. 2005

Talmud, P.J., Palmen, J., Putt, W., Lins, L., and Humphries, S.E.

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM®-7Zf Vector. Transcription/translation experiments were performed using the TNT® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ GreenLys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe® System –T7 and the Ribo m7G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

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Eukaryot. Cell 4, 1539-1549. Dual-luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae. 2005

McNabb, D.S., Reed, R., and Marciniak, R.A.

Notes: The firefly and Renilla luciferase coding regions were amplified from the pGL3 and pRL-CMV Vectors and cloned into various yeast expression vectors. Strains of Saccharomyces cerevisiae were transformed with these constructs and analyzed with the Dual-Luciferase® Reporter Assay System. The authors created yeast lysates for the Dual-Luciferase® Reporter Assay System using 1X Passive Lysis Buffer. Several factors important to assay performance as well as firefly and Renilla luciferase expression were explored, including the stability of both luciferases stored in lysates at room temperature for various periods of time, optimal culture density before lysis of transformants and the firefly luciferase half-life in S. cerevisiae. (3298)

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Plant Biotechnol. 22, 151–5. Multi-color luciferases as reporters for monitoring transient gene expression in higher plants 2005

Ogura, R., Matsuo, N., Wako, N., Tanaka, T., Ono, S. and Hiratsuka, K.

Notes: The authors evaluated use of a dual-color reporter assay for plant gene expression using the red and green light-emitting click beetle luciferase genes from the Chroma-Luc™ vectors (pCBR-Basic Vector, pCBG99-Basic Vector and pCBG68-Basic Vector). Plant expression vectors were constructed based on the reporter plasmid, pBI221, which contains the CaMV35S promoter, GUS reporter and nos-terminator cassette. The GUS cassette was replaced by the CBRluc, CBF99luc and CBR68luc genes. Twenty-five microliters of the gold microcarrier (1.6 mm) coated with 2µg plasmid DNA and were used to bombard plant specimens. Six hours after bombardment, an aqueous solution of 0.1mM D-luciferin potassium salt was sprayed on the transiently-tranfected plants and luminescence detected using a CCD camera system. For the color-specific detection, the researchers used interference filters for wavelengths greater than 610nm (high-pass filter) and wavelengths between 510nm and 560nm (band-pass filter). To test luciferase activity in cell extracts, cultured tobacco (BY-2) and onion epidermal cells were cotranfected by bombardment using the click beetle constructs and a plasmid with the 35S promoter driving Renilla luciferase. The cells were homogenized in Passive Lysis Buffer and 8µl of the prepared lysate was assayed using the Dual-Luciferase® Reporter Assay. A second plasmid containing the inducible CAB1 promoter from Arabidopsis thaliana, was made by replacing the firefly luciferase gene with the CBG99luc-coding sequence from the pCBG99-Basic Vector. Spinach leaves were transfected by bombardment with the CAB1 construct, exposed to different light conditions for 21 hours and luminescence levels detected by CCD camera and interference filters. In all cases, the authors were able to detect click beetle luciferase expression in plants and cultured cells. (3293)

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Mol. Cell. Biol. 25, 6031–46. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. 2005

Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang, Y.S., Lim, B., Robson, P. and Ng, H.H.

Notes: The authors studied the effects of Embryonic Stem Cell (ESC)-specific regulation on the Pou5f1 promoter in human and mouse cells. To examine the effect of knockdown of Oct4 and Sox2 (two genes involved in ESC regulation) on the Pou5f1 promoter, a 3kb fragment of the human POU5F1 promoter was cloned into pGL3-Basic Vector and 100ng cotransfected with 100ng shRNA plasmids into mouse E14 ESCs. Five nanograms of pRL-SV40 Vector served as a transfection control. For the enhancer assay, a 461bp fragment of genomic DNA containing the SRR2 enhancer of Sox2 was amplified and cloned into the pGL3-Promoter Vector. The same amounts of plasmid, shRNA and transfection control were transfected into E14 ESCs as in the Pou5f1 promoter assay. To investigate gene knockdown in 293T cells, 5ng of the two open reading frame (ORF) constructs (the Luc-Sox2 and the Luc-Pou5f1 ORFs cloned into the psiCHECK™-2 Vector) were cotransfected with 100ng shRNA plasmid. The outcome was examined 48–60 hours post-transfection using the Dual-Luciferase® Reporter Assay System. (3291)

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J. Biol. Chem. 280, 38029–38034. Regulation of Oxidative Stress by the Anti-aging Hormone Klotho 2005

Yamamoto, M., Clark, J.D., Pastor, J.V., Gurnani, P., Nandi, A., Kurosu, H., Miyoshi, M., Ogawa, Y., Castrillon, D.H., Rosenblatt, K.P. and Kuro-o, M.

Notes: Mice that overexpress Klotho exhibit an extended lifespan and delayed aging. In this study, the authors show that Klotho protein protects against oxidative stress and activates the FoxO transcription factors, inducing expression of manganese superoxide dismutase. Two luciferase constructs were made, one with luciferase under the control of the Fas ligand promoter and one under the control of the human SOD2 promoter. HeLa cells were transfected with one of the two luciferase constructs and the pRL-CMV Renilla control vector. Transfected cells were treated with or without Klotho protein, and cell lysates were analyzed using the Dual-Luciferase® Assay. Klotho protein stimulated the activity of the Fas ligand gene promoter and the SOD2 promoter. (3667)

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Blood 105, 4685-4692. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism. 2005

An, H., Xu, H., Zhang, M., Zhou, J., Feng, T., Qian, C., Qi, R. and Cao, X.

Notes: The authors of this study investigated the role of Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response. RAW264.7 macrophages were transfected with wildtype or mutant SHIP1 or control plasmid. Anti-ACTIVE® JNK and p38 antibodies were used in Western analyses to determine phosphorylation of these kinases in response to overexpression of SHIP1 or mutant SHIP1. To investigate any effects on IκB-alpha and NF-κB expression, RAW264.7 macrophages were cotransfected with pGL3-XκB-luciferase reporter plasmid and pRL-TK Renilla luciferase control plasmid. Transfected cells were treated with LPS for 6 hours or left untreated (control). The Dual-Luciferase® Reporter Assay System was used to monitor reporter gene expression. (3524)

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Nucl. Acids Res. 33, 4285-4310. Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for viral infectivity. 2005

Goffin, V., Demonté, D., Vanhulle, C., de Walque, S., de Launoit, Y., Burny, A., Collette, Y., Van Lint, C.

Notes: A fragment containing HIV-1 LAI 5' LTR was cloned into the unique EcoICRI-XhoI site of the pGL3-Basic Reporter Vector. The Luciferase Reporter Assay was used to analyze DNA transfected cells for luciferase activity. The pRL-TK Vector was used as a transfection efficiency internal control with a Renilla cDNA under control of HSV-TK. Firefly luciferase activity derived from the HIV-1 LTR was normalized to the Renilla luciferase activities using the Dual-Luciferase® Reporter Assay. (3703)

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J. Nutr. 135, 2987S-2992S. Zingiberaceous and citrus constituents, 1'-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action. 2005

Murakami, A., Shigemori, T. and Ohigashi, H.

Notes: These authors investigated the mechanisms by which anti-carcinogenic compounds derived from Japanese and subtropical vegetables and fruits attenuate LPS-induced COX-2 mRNA expression in RAW264.7 mouse macrophages. Using Western blot analysis, 10µg nuclear or 20µg cytosolic protein fractions isolated from LPS-treated macrophages in the absence or presence of various phytochemicals were stained with several antibodies for signal pathway proteins, including the Anti-ERK1/2 pAb. Further analysis of the MAPK and NF-κB systems was performed using firefly luciferase constructs co-transfected with the control pRL-TK Vector at a 1:1 ratio. Transfected cells were exposed to the plant compound for 12 hours and then to LPS for a further 12 hours. Reporter activity was measured using the Dual-Luciferase® Reporter Assay System. To determine the effect of the phytochemical zerumbone on the kinase reaction, a cell-free kinase assay was performed using Kinase-Glo® Assay System. In this assay, 1.5µl recombinant MAPKAPK-2, 0.2µl recombinant active p38α, 1µl zerumbone and 4µmol/l ATP were incubated for 3 hours at 28°C before addition of an equal volume of Kinase-Glo® Reagent. (3333)

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J. Biol. Chem. 279(10), 8787-8791. 14-3-3beta binds to big mitogen-activated protein kinase 1 (BMK1/ERK5) and regulates BMK1 function. 2004

Zheng Q., Yin G., Yan C., Cavet M. and Berk B.C.

Notes: The authors performed a yeast two-hybrid screen using big mitogen-activated kinase 1 (BMK1/ERK5) as the bait and identified the scaffolding protein 14-3-3beta. To confirm this interaction, the cloned mouse BMK1 gene was expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The expressed protein was labeled with Transcend™ tRNA.  Using a GST-14-3-3beta fusion protein, a pull-down assay was performed and the direct binding confirmed after immunoblotting and staining with streptavidin-horseradish peroxidase (HRP).  The interaction of various BMK1 mutants were tested in a mammalian two-hybrid system and measured by the Dual-Luciferase® Reporter Assay System. (3078)

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Proc. Natl. Acad. Sci. USA 97(12), 6597-6602. Adaptive variation in lactate dehydrogenase-B gene expression: role of a stress-responsive regulatory element. 2004

Schulte, P.M., Glemet, H.C., Fiebig, A.A. and Powers, D.A.

Notes: The research in this article describes differences in the regulatory region of the lactate dehydrogenase-B (Ldh-B ) gene between isolates of Fundulus heteroclitus. To study Ldh-B regulation, 5’ regulatory sequences were cloned from isolates that live at different temperatures. These sequences were subsequently cloned upstream of the firefly luciferase gene. Between 10-60μg of this construct and 5μg of pRL-CMV Vector in physiological saline were injected directly into the liver of the fish using tempera paint as an injection indicator. Seven days after injection, the livers were removed and homogenized with a Polytron homogenizer in Passive Lysis Buffer. The lysate was cleared twice at 10,000 x g for 15 minutes and then stored frozen for 24 hours before being used in the Dual Luciferase® Assay. (3068)

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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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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.


(4361)

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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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