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Cancer Res. 67, 1239–1245. TSP50 encodes a testis-specific protease and is negatively regulated by p53. 2007

Xu, H., Shan, J., Jurukovski, V., Yuan, L., Li, J. and Tian, K.

Notes: TSP50 is a testis-specific gene found to be overexpressed in human breast cancer tissue. Of interest is a putative p53 binding site in the TSP50 promoter. To examine what effect p53 may have on TSP50 expression, the TSP50 promoter was cloned into a pGL3 Luciferase Reporter Vector and cotransfected with a wildtype or R249S mutant p53 and a control vector, pCMV/β-galactosidase, into HeLa, HEK293 and paired MCF7 cells. After 24 hours, the cells were assessed for luciferase expression using the Luciferase Assay System and normalized to β-galactosidase expression, which was measured using the Beta-Glo® Assay System. (3598)

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Mol. Cell. Biol. 27, 7947-7954. Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene. 2007

Igarashi, M., Yogiashi, Y., Mihara, M., Takada, I., Kitagawa, H. and Kato, S.

Notes: Igarashi et al. showed that expression of the Msx2 gene, which enodes a transcription factor, is induced by vitamin K treatment via a pregnane X receptor response element (PXRE) and by estrogen via an estrogen response element (ERE). Promoter analysis was performed by cloning the Msx2 promoters into the pGL3-Basic Vector, transfecting MC3T3 and ST2 cells with the pGL3-Basic constructs, treating the cell with 10nM 17β-estradiol and 10µM vitamin K, then measuring luciferase activity. The pRL-CMV Vector (2.5ng per well of a 12-well plate) was cotransfected to normalize for transfection efficiency. The ability of PXR to bind to the Msx2-PXRE was assessed by an avidin-biotin complex DNA assay. Sense and antisense oligonucleotides that were biotinylated at the 5´ end were annealed and immobilized with the TetraLink™ Tetrameric Avidin Resin. HEK293T cells were lysed with lysis buffer (10mM Tris-HCl [pH7.8], 1mM EDTA, 150mM NaCl, 0.1% NP-40) containing protease inhibitors, then centrifuged to clarify the extract. The supernatants were mixed with the DNA-TetraLink™ Resin to allow proteins to bind to the oligos, and resin was washed with lysis buffer. Bound proteins were analyzed by SDS-PAGE and Western blotting. (3805)

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J. Leukoc. Biol. 79, 628-638. Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages. 2006

Basler, T., Jeckstadt, S., Valentin-Weigand, P., and Goethe, R.

Notes: In this study a 984bp fragment of the IRG1 5´ promoter region was cloned into the pGL3 Basic Vector. Transfection-quality plasmid DNA was purified using the PureYield™ Plasmid Midiprep System and used to transfect RAW264.7 cells. Twenty-four hours post-transfection, cells were stimulated with LPS or infected with Mycobacterium paratuberculosis or Mycobacterium smegmatis for an additional 24 hours. Relative luciferase activities in LPS-stimulated and infected macrophages were then assayed using the Dual-Luciferase® Reporter Assay System. (3365)

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Proc. Natl. Acad. Sci. USA 103, 4552-4557. A dopamine transporter gene functional variant associated with cocaine abuse in a Brazilian sample. 2006

Guindalini, C., Howard, M., Haddley, K., Laranjeira, R., Collier, D., Ammar, N., Craig, I., O'Gara, C., Bubb, V.J., Greenwood, T., Kelsoe, J., Asherson, P., Murray, R.M., Castelo, A., Quinn, J.P., Vallada, H., and Breen, G.

Notes: These authors investigated the effect of various polymorphisms in the dopamine transporter gene (SLC6A3) on susceptibility to cocaine addiction. Genotyping of various polymorphisms in cocaine abusers and control subjects revealed a potential association of the int8 VNTR with cocaine abuse. Seven alleles of the int8 VNTR were sequenced. Various allelic sequences were then cloned into a modified phRL-SV40 Renilla luciferase reporter vector and transfected into the mouse SN4741 cell line, which expresses the dopamine transporter, and the effects on reporter activity were monitored. Sequences of two alleles were then cloned into a pGL3 Promoter Vector construct and transfected into JAP cells. The cells were then challenged with various amounts of cocaine, KCL or KCl and forskolin, and the effect on reporter activity was monitored. The TransFast™ Reagent was used for transfections at a 2:1 reagent:DNA ratio. (3543)

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J. Clin. Oncol. 24, 983-7. Elevated serum B-lymphocyte stimulator levels in patients with familial lymphoproliferative disorders. 2006

Novak, A.J., Grote, D.M., Ziesmer, S.C., Kline, M.P., Manske, M.K., Slager, S., Witzig, T.E., Shanafelt, T., Call, T.G., Kay, N.E., Jelinek, D.F., Cerhan, J.R., Gross, J.A., Harder, B., Dillon, S.R. and Ansell, S.M.

Notes: To test the significance of the C or T polymorphism at position -871 of the serum B-lymphocyte stimulator (BLyS) promoter region, the BLyS promoter was amplified, digested with Kpn I and cloned into the pGL3-Enhancer Vector. HL60 cells were then transiently transfected by electroporation using 10µg of the pGL3-BLyS-promoter constructs and 40ng of the pGL4.75[hRluc/CMV] Vector, which expresses Renilla luciferase. After 48 hours, expression of the reporter genes was assessed using the Dual-Luciferase® Reporter Assay System. (3343)

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FEBS Lett. 580, 755-762. Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). 2006

Lee, S.J., Jang, B.C., Lee, S.W., Yang, Y.I., Suh, S.I., Park, Y.M., Oh, S., Shin, J.G., Yao, S., Chen, L. and Choi, I.H.

Notes: Many cancer cells upregulate the co-signaling molecule B7-H1, confering resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System. (3451)

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Cancer Res. 66, 9090-9098. MicroRNA regulates the expression of human cytochrome P450 1B1. 2006

Tsuchiya, Y., Nakajima, M., Takagi, S., Taniya, T., and Yokoi, T.

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

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J. Biol. Chem. 281, 2044–2052. Proline oxidase, a proapoptotic gene, is induced by troglitazone: evidence for both peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms. 2006

Pandhare, J., Cooper, S.K. and Phang, J.M.

Notes: A proline oxidase (POX) antisense vector was generated by amplifying part of the POX cDNA and ligating the product into the pCI Mammalian Expression Vector in the antisense orientation. This construct was tested and validated for blocking POX mRNA expression using RT-PCR. Both PPARγ and p53 cDNAs were also cloned into the pCI Vector. The human POX promoter sequence was amplified and cloned into the NheI and HindIII sites of the pGL3-Basic Vector to create the POX-Luc reporter construct. Using several colon cancer cell lines (HT29, LoVo, HCT116, HCT15, RKO, KM12, HCC2998 and SW620), the POX-Luc construct was co-transfected with pRL-null (to normalize transfection efficiency) plus PPARγ, p53 contructs or empty vector. A PPARγ ligand was added 10 hours post-transfection and cells harvested 24–36 hours after transfection. POX promoter luciferase activity was measured using the Dual-Luciferase® Reporter Assay System and a TD-20/20 luminometer. (3514)

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J. Biol. Chem. 280, 28412-28423. Protein Kinase C βII plays an essential role in dendritic cell differentiation and autoregulates its own expression. 2006

Cejas, P.J., Carlson, L.M., Zhang, J., Padmanabhan, S., Kolonias, D., Lindner, I., Haley, S., Boise, L.H. and Lee, K.P.

Notes: Protein Kinase C activity was assayed in unstimulated KG1, KG1a, KG1a-neo and KG1a-PKC-βII-GFP human leukemic cells using the SignaTECT® Protein Kinase C (PKC) Assay System. For PKC-βII promoter analysis, reporter constructs were cloned into the pGL3-Basic Vector. The pRL-CMV plasmid was used as an internal control to normalize luciferase activity. Reporter assays were carried out using the Dual-Luciferase® Reporter Assay System. (3407)

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J. Immunol. 176, 5519-5528. Reduced nitric oxide synthase 2 (NOS2) promoter activity in the Syrian hamster renders the animal functionally deficient in NOS2 activity and unable to control an intracellular pathogen. 2006

Perez, L.E., Chandrasekar, B., Saldarriaga, O.A., Zhao, W., Arteaga, L.T., Travi, B.L. and Melby, P.C.

Notes: Leishmania donovani infection elicits an immune response in mice macrophages that includes the upregulation of nitric oxide synthase 2 (NOS2). Hamster and human macrophages do not exhibit an upregulation of NOS2 upon infection. The authors measured the activities of the NOS2 promoter in response to interferon-γ (IFNγ) and lipopolysaccharide treatment of mouse, hamster and human macrophages. The mouse, hamster and human NOS2 promoters were cloned into pGL3-Basic Vector and transfected into mouse macrophages by electroporation. Promoter activities were determined using the Dual Luciferase® Reporter Assay System. The pRL-null Vector was used to normalize for differences in transfection efficiency (3470)

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J. Immunol. 176, 5050–5059. Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells. 2006

Koon, H.W., Zhao, D., Zhan, Y., Rhee, S.H., Moyer, M.P. and Pothoulakis, C.

Notes: To examine the effect of Substance P (SP) on COX-2 expression, the COX-2 promoter region spanning –2069 to –66 bp was cloned by PCR and subcloned into the pGL3-Basic Vector (pGL3-Cox-2). Nontransformed human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NK-1R; NCM460-NK-1R) were seeded in 12-well plates and transiently transfected with pGL3-Cox-2 with either a transfection control pRL-TK Vector or siRNA or both. The siRNA molecules used were for AK1, JAK2 (Upstate Biotechnology), STAT3, STAT5, STAT6 or a control siRNA. The transfected cells were serum starved for 24 hours, treated with SP for 4 hours and then lysed. The cell extracts were measured for firefly and Renilla luciferase activities using the Dual-Luciferase® Reporter Assay System. Relative luciferase activity was a ratio of COX-2 promoter firefly activity to Renilla activity; results were expressed as percentage of control group without SP stimulation. To mutate the STAT binding elements, the pGL3-Cox-2 construct was modified using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting mutant constructs were tested in the same system as the wildtype COX-2 promoter. (3520)

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J. Immunol. 176, 5050-9. Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells. 2006

Koon, H-W., Zhao, D., Zhan, Y., Rhee, S.H., Moyer, M.P. and Pothoulakis, C.

Notes: The COX-2 promoter was cloned by PCR into the pGL3 Vector. NCM460-NK-1R cells were transiently transfected with the cloned promoter and the pRL-TK vector as an internal control or siRNA targeted against various JAK/STAT genes. The Dual-Luciferase® Assay was used to measure promoter activity. The wildtype COX-2 promoter was mutagenized using the GeneEditor™ in vitro Site-Directed Mutagenesis System. (3384)

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J. Biol. Chem. 281, 17635–17643. The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter. 2006

de Wolf, C.J., Cupers, R.M., Bertina, R.M. and Vos, H.L.

Notes: The Protein S promoter (PROS1) fragment –5948/–1 was cloned directly 5’ to the firefly luciferase reporter gene in the pGL3-Basic Vector using the KpnI and XhoI enzyme sites. This construct, PS5948-luc, was linearized with KpnI and NdeI and subsequently subjected to progressive deletion using the Erase-a-Base® System. The size of the resulting 5’-deletion was determined by sequence analysis, and the deletion constructs were used for transient transfection assays. HepG2, HuH7, HeLa and HUVEC cells were transfected at 60–80% confluency in 12-well plates using 3µl of Tfx™-20 per microgram DNA. In each transfection, an equimolar concentration of construct was used and supplemented with an additional plasmid to keep the amount of transfected DNA constant. pRL-SV40 Vector was co-transfected as a transfection control using a 1:500 ratio to the total transfected amount of DNA in HepG2, HuH7 and HeLa cell lines, and a 1:100 ratio in transfections with HUVEC and 1 × 106 Meg01 suspension cells. Transcription factor expression vector (250ng) was co-transfected, and expression vector without the transcription factor cDNA was used as a negative control. Cell extracts were harvested at either 24 (HepG2 and HuH7) or 48 hours (Meg01, HUVEC, and HeLa) post-transfection using 250µl of Passive Lysis Buffer per well. Luciferase activity was determined using 20–100µl of lysate with the Dual-Luciferase® Reporter Assay System. (3510)

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J. Biol. Chem. 281, 9030-9037. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. 2006

Ullah, M.S., Davies, A.J. and Halestrap, A.P.

Notes: Monocarboxylate transporters (MCT) transport lactic acid across the cell membrane. The promoters of 4 MCT family members (MCT1, MCT2, MCT3 and MCT4), were amplified by PCR and cloned into the pGEM®-T Easy Vector. The sequences were confirmed, and the promoters were cloned into the pGL3-Basic Vector. The Dual-Glo™ Luciferase Assay System was used to quantitate promoter activity under basal and hypoxic conditions in HeLa cells. The pRL-SV40 Vector was used to normalize for differences in transfection efficiency. (3463)

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Drug Metab. Dispos. 33, 1244–53. Sp1 and Sp3 regulate basal transcription of the human CYP2F1 gene. 2005

Wan, J., Carr, B.A., Culter, N.S., Lanza, D.L., Hines, R.N. and Yost, G.S.

Notes: The human lung cell line A549 and the human liver cell line were transiently transfected with 0.1µg of CYP2F1 reporter constructs and 0.001µg of pRL-TK Vector using FuGENE® 6 Reagent in 96-well plates. For cotransfection studies, cells were transfected with 0.1µg of the reporter construct, 0.002µg of pRL-TK plasmid, and 0.5 or 0.2µg of Sp1, Sp3 or empty expression vectors, with the total transfected DNA remaining at 0.2µg. Reporter activity was assessed using the Dual-Luciferase® Reporter Assay System.

SL-2 cells were seeded in 24-well plates and cotransfected with 0.3µg of CYP2F1 reporter plasmid and 0.3µg of pPac/Sp1, pPac/Sp2 or empty expression vector. The total amount of plasmid DNA used for each transfection was 0.9µg. The DNA and FuGENE® 6 were added at a 3:1 ratio. Activities were assessed using the Dual- Luciferase® Reporter Assay System.

The Gel Shift Assay System was used to identify Sp1-like sites in the promoter of the human CYP2F1 using EMSA (electromobility shift analysis). (4269)

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J. Biol. Chem. 280, 19977-19985. A novel Myc-target gene, mimitin, that is involved in cell proliferation of esophageal squamous cell carcinoma. 2005

Tsuneoka, M., Teye, K., Arima, N., Soejima, M., Otera, H., Ohashi, K., Koga, Y., Fujita, H., Shirouzu, K., Kimura, H. and Koda, Y.

Notes: The authors used 5´ and 3´RACE to amplify the gene mimitin, and the resulting cDNA was cloned into the pGEM®-T Vector. A genomic DNA fragment containing the mimitin promoter sequence was amplified by PCR and cloned into the pGEM®-T Vector. The promoter was then cloned into the pGL3-Basic Vector. The activity of the wildtype and mutated promoters was determined using a luciferase assay. The pRL CMV Vector was used to normalize for differences in transfection efficiency. (3466)

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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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Cancer Res. 65, 10024–10031. Epigenetic regulation of WTH3 in primary and cultured drug-resistant breast cancer cells. 2005

Tian, K., Jurukovski, V., Wang, X-P., Kaplan, M.H. and Xu, H.

Notes: Methylation of the WTH3 promoter region has been associated with multidrug resistance in breast cancer cells in vitro. In this study, the effect of WTH3 in primary cultured multidrug resistant cells was evaluated, and the influence of a frequently methylated CpG23 region on WTH3 promoter activity was investigated using a luciferase reporter assay system. Promoter regions containing wildtype or mutated CpG regions were cloned into the pGL3 Vector and transfected into paired MCF7 cell lines, along with a control pGL3 Vector without insert, and a plasmid containing the β-galactosidase gene as a transfection efficiency control. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System, and β-galactosidase activity was determined using the Beta-Glo® Assay System. Luciferase activities of the wildtype and mutant promoter regions were compared after normalization to β-galactosidase activity and protein concentration. (3457)

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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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Nucl. Acids Res. 33, 4140–56. Functional polarity is introduced by Dicer processing of short substrate RNAs. 2005

Rose, S.D., Kim, D.H., Amarzguioui, M., Heidel, J.D., Collingwood, M.A., Davis, M.E., Rossi, J.J. and Behlke, M.A.

Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells. A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

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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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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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Proc. Natl. Acad. Sci. USA 101(9), 2794-2799. Differential utilization of upstream AUGs in the beta-secretase mRNA suggests that a shunting mechanism regulates translation. 2004

Rogers G.W. Jr, Edelman G.M. and Mauro V.P.

Notes: The authors studied the behavior of the three upstream AUGs to the initiation codon of beta-Secretase (BACE1) in a cell-free expression system to understand how this 5´ upstream region affects translation.  The 5´ UTR (with or without a hairpin structure) was cloned in front of the firefly luciferase gene derived from the pGL3-Control Vector. The resultant construct was either transfected into cells or in vitro transcribed to yield capped or uncapped RNA followed by translation in Rabbit Reticulocyte Lysate, Nuclease Treated, with or without m7GpppG cap analogue.  The experiment demonstrated that both the hairpin structure and the 5´ cap analogue decreased the translation level of the BACE1 protein.  (3076)

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J. Immunol. 172, 5512–5521. Human CD1D gene has TATA boxless dual promoters: An SP1-binding element determines the function of the proximal promoter. 2004

Chen, Q.Y. and Jackson, N.

Notes: These authors demonstrated that the human CD1D gene has distal and proximal TATA boxless promoter sequences. Distal and proximal promoters to CD1D were cloned into the pGL3-Basic Vector to create reporter constructs. One construct contained the entire 4,986 base pair region containing distal and proximal CD1D promoter.  Transient transfections were performed using 5 x 105 Jurkat cells in 24-well plates, 0.8 μg of pGL3 Basic Vector with the insert of interest, and 30ng of pRL-CMV Vector as a transfection normalization control. The Dual-Glo™ Luciferase Assay System was used to assay luciferase activities.  (3061)

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