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| 1. |
Kizaka-Kondoh, S., Itasaka, S., Zeng, L., Tanaka, S., Zhao, T., Takahashi, Y., Shibuya, K., Hirota, K., Semenza, G.L. and Hiraoka, M.
(2009)
Selective killing of hypoxia-inducible factor-1-actie cells improves survival in a mouse model of invasive and metastatic pancreatic cancer
Clin. Can. Res.
15
,
3433-3441
.
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| |
Notes:
Hypoxia within solid tumors is a major factor that contributes to cancer progression. Pancreatic cancers express target genes of the transcription factor hypoxia-inducible factor-1. These genes encode proteins involved in angiogenesis, such as vascular endothelial growth factor (VEGF) and other growth factors like insulin-like growth factor (IGF) and proteins associated with the extracellular matrix. The authors of this study investigated whether HIF-1 activity plays a role in progression of pancreatic cancers. They implanted the human pancreatic cancer cells (SUIT-2) that had been transfected with a luciferase reporter in to nude male mice. Mice were either untreated or treated with the prodrug POP33, which appears to increase capsase-3 activity. They sought to determine if POP33 would induce apoptosis in HIF-1 expressing hypoxic cells. Bioluminescent imaging (BLI) was performed in vivo by injecting mice with D-luciferin solution. Caspase activity was also monitored in vivo using a luminescent caspase-3/7 substrate.
(0003997) |
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Products: VivoGlo™ Caspase-3/7 Substrate (Z-DEVD-Aminoluciferin) |
| 2. |
Hornakova, T., Staerk, J., Royer, Y., Flex, E., Tartaglia, M., Constantinescu, S.N., Knoops, L. and Renauld, J.C.
(2009)
Acute lymphoblastic leukemia-associated JAK1 mutants activate the Janus kinase/STAT pathway via interleukin-9 receptor alpha homodimers.
J. Biol. Chem.
284
,
6773–6781
.
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| |
Notes:
The authors studied the ability of JAK1 V658F and A634D mutants to activate the Janus kinase (JAK)/STAT pathway when expressed alone or together with the other components of the interleukin-9 receptor complex. The BOX1 motif of wild-type IL-9Rα, the JAK interacting region, was mutated from PXP to SXS using the GeneEditor™ in vitro Site-Directed Mutagenesis System. To assess STAT transcriptional activity, HEK293 human embryonic kidney, COS-7 monkey kidney, U4C human fibrosarcoma and g2A cells were cotransfected with 250ng of the appropriate constructs, 500ng of firefly luciferase vectors and 50ng of pRL-TK Vector and empty plasmid for a total 1.5µg of DNA. After 24 hours, the cells were lysed in 150µl of Passive Lysis Buffer and reporter activity measured using the Dual-Luciferase® Reporter Assay System. The ProFection® Mammalian Transfection System—Calcium Phosphate was used to transfect 106 HEK293 cells in a six-well plate with 3.75µg of plasmid for Western blot analysis and cotransfected 6 × 106 HEK293 cells in a 100mm dish with 14µg plasmid for immunoprecipitation studies.
(0004025) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | GeneEditor™ in vitro Site-Directed Mutagenesis System | pRL-TK Vector | ProFection® Mammalian Transfection System—Calcium Phosphate |
| 3. |
Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.
(2009)
Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage.
Nucleic Acids Res.
37
,
78–95
.
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Notes:
The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 103 cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter® 96 AQueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System.
(0004031) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CellTiter 96® AQueous One Solution Reagent | CellTiter 96® AQueous One Solution Reagent | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Gel Shift Assay Core System | Gel Shift Assay System | GeneEditor™ in vitro Site-Directed Mutagenesis System | pCI Mammalian Expression Vector | pGL3 Basic Vector | Plexor® qPCR System | pRL-TK Vector |
| 4. |
Zheng, Y., Ritzenthaler, J.D., Sun, X., Roman, J. and Han, S.
(2009)
Prostaglandin E2 stimulates human lung carcinoma cell growth through induction of integrin-linked kinase: the involvement of EP4 and Sp1.
Cancer Res.
69
,
896–904
.
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Notes:
In this paper, the role of prostaglandin E2 (PGE2) stimulation of integrin-linked kinase (ILK) in human lung carcinoma was explored. Mutations of Sp1 and NF-κB cis-acting elements in an ILK promoter-pGL3-Basic Vector construct were created using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The mutations were confirmed via sequencing. Human non–small cell lung carcinoma (NSCLC) cells were plated at a density of 5 × 105 cells per well in six-well plates and transfected with 2µg of ILK promoter reporter vectors with or without 0.2µg of the phRL-TK Renilla Luciferase Reporter Vector. After 24 hours, the transfected cells were exposed to PGE2 and the cells lysed for assessment using the Dual-Luciferase® Reporter Assay System. NSCLC cells were transfected with inactive (ILK-S343A) and superactive ILK (ILK-S343D) cDNA, incubated for 24 hours, treated with or without exogenous PGE2 or with an Sp1 inhibitor for 2 hours. The numbers of viable cells were measured using the CellTiter-Glo® Luminescent Cell Viability Assay.
(0004026) |
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Products: CellTiter-Glo® Luminescent Cell Viability Assay | Dual-Glo® Luciferase Assay System | GeneEditor™ in vitro Site-Directed Mutagenesis System | pGL3 Basic Vector |
| 5. |
Rahmani, F., Hummel, M., Schuurmans, J., Wiese-Klinkenberg, A., Smeekens, S. and Hanson, J.
(2009)
Sucrose control of translation mediated by an upstream open reading frame-encoded peptide.
Plant Physiol.
150
,
1356–1367
.
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Notes:
The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER® Vector and amino acid substitutions were introduced using the Altered Sites® II in vitro Mutagenesis System. The pGEM®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase® Reporter Assay System.
(0004023) |
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Products: Altered Sites® II in vitro Mutagenesis System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pALTER®-1 Vector | pGEM®-T Vector | pGEM®-T Vector System I | pGEM®-T Vector System II |
| 6. |
Binkowski, B., Fan, F., Wood, K.
(2009)
Engineered luciferases for molecular sensing in living cells
Curr. Opin. Biotechnol
20
,
14-8
.
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Notes:
Sophisticated tools are required to gain insight into the complicated mileau that makes up intracellular machinery. One means of gaining this insight is creation of intracellular biosensors using structurally engineered reporter proteins as biosensors. These biosensors can serve as probes within living cells, transmitting a signal regulated by the sensor is regulated by its interaction with cellular components. This article highlights the strategies behind a number of designs that have been described for preparing luciferase-containing intramolecular sensors.
(0004009) |
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Products: pCBG99-Control Vector | pCBR-Control Vector |
| 7. |
Emonet, S.F., Garidou, L., McGavern, D.B. and de la Torre, J.C.
(2009)
Generation of recombinant lymphocytic chriomeningits virus with trisegmented genomes stably expressing two additional genes of interest.
Proc. Natl. Acad. Sci. USA
106
,
3473-3478
.
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Notes:
The lymphocytic choriomeningitis virus (LCMV) was used as a model to create a trisegmented recombinant arenavirus in which viral genes were replaced by a gene of interest. One such engineered virus, r3LCMV CAT/FLuc, was used in a pilot screen to identify anti-arenaviral compounds. Firefly luciferase (FLuc) activity was measured using the ONE-Glo™ Luciferase Assay System.
(0003957) |
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Products: ONE-Glo™ Luciferase Assay System |
| 8. |
Brunzell, D.H., Mineur, Y.S, Neve, R.L. and Picciotto, M.R.
(2009)
Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference.
Neuropsychopharmacology
Feb. 11
,
(epub ahead of print)
.
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| |
Notes:
The authors of this study used the HRE-CRE-luciferase reporter cell line (Glo-Response™ Cells) to test HSV constructs for activity. Cells were infected with HSV-CREB, HSV-mCREB (dominant negative) or HSV-LacZ control vector. Comparisons indicated that cells transfected with HSV-CREB showed increase in CRE-mediated activity, while those transfected with HSV-mCREB showed attenuation of CRE-mediated cellular activity.
(0003956) |
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Products: GloResponse™ CRE-luc2P HEK293 Cell Line |
| 9. |
Shigemoto, T., Kageyama, M., Hirai, R., Zheng, J., Yoneyama, M. and Fujita, T.
(2009)
Identification of loss of function mutations in human genes encoding RIG-I and MDA5: implications for resistance to type I diabetes.
J. Biol. Chem.
284
,
13348–13354
.
|
| |
Notes:
Here the authors studied various non-synonymous SNPs of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) that are essential for detecting viral RNA and triggering antiviral responses. Various point mutations were introduced into RIG-1 and MDA5 using the GeneEditor™ in vitro Site-Directed Mutagenesis System with pEF-FLAG clones. Mouse embryonic fibroblasts (MEFs) and L929 cells were cotransfected with RIG-I mutants or MDA5mutants and pRL-TK Vector, and stimulated with RNA or viral infection. Reporter activity was measured using the Dual-Luciferase® Reporter Assay System.
(0004024) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | GeneEditor™ in vitro Site-Directed Mutagenesis System | pRL-TK Vector |
| 10. |
Cho, Y.Y., Tang, F., Yao, K., Lu, C., Zhu, F., Zheng, D., Pugliese, A., Bode, A.M. and Dong, Z.
(2009)
Cyclin-dependent kinase-3-mediated c-Jun phosphorylation at Ser63 and Ser73 enhances cell transformation.
Cancer Res.
69
,
272–281
.
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| |
Notes:
This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 103 in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter® 96 AQueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected.
(0004028) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CheckMate™ Mammalian Two-Hybrid System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pACT Vector | pBIND Vector | pG5luc Vector |
| 11. |
Xia, M., Huang, R., Guo, V., Southall, N., Cho, M.H., Inglese, J., Austin, C.P. and Nirenberg, M.
(2009)
Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory.
Proc. Natl. Acad. Sci. U S A
106
,
2412–7
.
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Notes:
In this study, small molecule enhancers of cAMP response element binding (CREB) were studied using quantitative high-throughput screening. After an initial screen of 73,000 compounds, 1,800 compounds were classified as potentiators of CREB activity. A second screening to confirm the compound potential was performed using the GloResponse™ CRE-luc2P HEK293 Cell Line. Five microliters of cells in assay medium were seeded in 1,536-well plates at a density of 2,500 cells/well. The next day, 23 nl of compound in DMSO or DMSO alone was dispensed into each well, then 1 μl of NKH477 (final concentration, 200 nM) or media alone was added to the assay plates. After incubating the cells for 4 hours at 37 °C, 6 μl of Bright-Glo™ Luciferase Assay Reagent was added to each well, incubated at room temperature for 10 minutes and the luminescence measured.
(0004004) |
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Products: Bright-Glo™ Luciferase Assay System | GloResponse™ CRE-luc2P HEK293 Cell Line |
| 12. |
Mie, M., Shimizu, S., Takahashi, F. and Kobatake, E.
(2008)
Selection of mRNA 5´-untranslated region sequence with high translation efficiency through ribosome display.
Biochem. Biophys. Res. Commun.
373
,
48–52
.
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Notes:
The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency.
(0003963) |
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Products: Renilla Luciferase Assay System | Flexi® Rabbit Reticulocyte Lysate System | MagneHis™ Ni-Particles | TNT® T7 Coupled Reticulocyte Lysate System |
| 13. |
Shibuya, N., and Nakashima, N.
(2008)
Characterization of the 5' internal ribosome entry site of Plautia stali intestine virus.
J. Gen. Virol.
87
,
3679-3686
.
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Notes:
The Plautia stali virus contains two open reading frames and includes a 5´ internal ribosome entry site (IRES) and an intergenic IRES region. These authors showed that the 5´ IRES was functional and initiated translation in insect cell lysate, but not in rabbit reticulocyte lysate or wheat germ extract. The efficiency of translation mediated by the 5´ IRES region was tested with and without cap analogue using various firefly and Renilla luciferase reporter constructs. They also used deletion mutants to identify the specific regions required for translation initiation.
(0003942) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System | pSP-luc+NF Fusion Vector | Transcend™ tRNA |
| 14. |
Kazeto, Y., Kohara, M., Miura, T., Miura, C., Yamaguchi, S., Trant, J.M., Adachi, S. and Yamauchi, K.
(2008)
Japanese eel follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): production of biologically active recombinant Fsh and Lh by Drosophila S2 cells and their differential actions on the reproductive biology.
Biol. Reprod.
79
,
938–946
.
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Notes:
The ORFs of Japanese eel follicle-stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) were ligated into the pSI Mammalian Expression Vector. Fifty nanograms of this construct or the pSI Vector was cotransfected with 0.5ng of pRL-null Vector and 1µg or a cAMP-responsive firefly luciferase reporter vector into COS cells seeded in 60mm dishes. The next day, the cells were trypsinized, replated into 96-well plates and allowed to recover for 1 day. The transfected cells were serum-starved then treated with hormones six hours before being lysed and enzymatic activity measured with the Dual-Luciferase® Reporter Assay System.
(0003988) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pRL-null Vector | pSI Mammalian Expression Vector |
| 15. |
Purbey, P.K., Singh, S., Kumar, P.P., Mehta, S., Ganesh, K.N., Mitra, D. and Galande, S.
(2008)
PDZ domain-mediated dimerization and homeodomain-directed specificity are required for high-affinity DNA binding by SATB1.
Nucleic Acids Res.
36
,
2107–2722
.
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Notes:
To learn about the ideal target binding sequence for SATB1, the T-lineage-enriched chromatin organizer and transcription factor, random oligonucleotides underwent SELEX and five rounds of selection by EMSA. The enriched library of oligos was cloned into the pGEM®-T Easy Vector, transformed and sequenced. Several variants of SATB1-binding consensus sequences were annealed, ligated into the pGL3-Promoter Vector and cotransfected into HEK 293 cells with a plasmid that either contained SATB1 or was empty. After 48 hours, the cells were harvested and luciferase activity measured. The CheckMate™ Mammalian Two-Hybrid System was used to assess how the N-terminal PDZ domain of SATB1 interacted with the Cut and homeodomain in the C-terminus.
(0003982) |
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Products: CheckMate™ Mammalian Two-Hybrid System | pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | pGL3-Promoter Vector |
| 16. |
Hawes, J.J., Nerva, J.D. and Reily, K.M.
(2008)
Novel dual-reporter preclinical screen for antiastrocytoma agents identifies cytostatic and cytotoxic compounds
J. Biolmol. Scr.
13
,
795-803
.
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Notes:
The authors of this paper created a mouse model for in vitro assays to screen for therapeutic compounds specifically active against astrocytic gliomas. A Green/Red luciferase (G/R-luc) dual-reporter system was created in KR158 cells (derived from grade III agressive mouse anaplastic astrocytoma). The green click beetle luciferase gene (from pCBC68-Basic Vector) was placed under the control of the E2F1 promoter, and the red click beetle luciferase gene (from pCBR-Basic Vector) was placed under the control of the CMV promoter. The dual-reporter assay simultaneously evaluates E2F1 promoter activity and assesses cytotocity; the assay also distinguishes cytostatic from cytotoxic compounds.
(0003949) |
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Products: Chroma-Glo™ Luciferase Assay System | pCBG68-Basic Vector | pCBR-Basic Vector |
| 17. |
Cali, J.J., Niles, A., Valley, M.P., O’Brien, M.A., Riss, T.L., and Shultz, J.
(2008)
Bioluminescent assays for ADMET
Expert Opin. Drug Metab. Toxicol.
4
,
103–120
.
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Notes:
The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase.
(0003926) |
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Products: Apo-ONE® Homogeneous Caspase-3/7 Assay | Apo-ONE® Homogeneous Caspase-3/7 Buffer | Beta-Glo® Assay System | Calpain-Glo™ Protease Assay | Caspase-Glo® 2 Assay | Caspase-Glo® 3/7 Assay | Caspase-Glo® 6 Assay | Caspase-Glo® 8 Assay | Caspase-Glo® 9 Assay | CellTiter-Glo® Luminescent Cell Viability Assay | GloResponse™ CRE-luc2P HEK293 Cell Line | GloResponse™ NFAT-RE-luc2P HEK293 Cell Line | GSH-Glo™ Glutathione Assay | Kinase-Glo® Luminescent Kinase Assay | Kinase-Glo® Max Luminescent Kinase Assay | Kinas |
| 18. |
Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.
(2008)
miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes.
Nucleic Acids Res.
36
,
5391–404
.
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Notes:
To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control.
(0003894) |
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Products: Dual-Luciferase® Reporter Assay System | ImProm-II™ Reverse Transcriptase | pGL3-Control Vector | pRL-TK Vector | T7 RiboMAX™ Express Large Scale RNA Production System |
| 19. |
Gerlach, R.G., Hölzer, S.U., Jäckel, D., and Hensel, M.
(2008)
Rapid engineering of bacterial reporter gene fusions by using Red recombination.
Appl. Environ. Microbiol.
73
,
4234-4242
.
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Notes:
These authors describe use of a red recombinase mediated method for generation of reporter constructs in Salmonella enterica setrovar typhimurium. Among the reporter constructs created was a HaloTag® reporter using the HaloTag® coding region from the pHT2 promoter.
(0003924) |
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Products: HaloTag® pHT2 Vector |
| 20. |
Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugris, G., Zhu, J., Darzins, A., Klaubert, D.H., Bulleit, R.F., and Wood, K.V.
(2008)
HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis
ACS Chemical Biology
3
,
373–382
.
|
| |
Notes:
The authors of this study describe a reporter gene system that allows researchers to create one genetic construct that can be used for a variety of studies including imaging and protein imobilization. The HaloTag® reporter protein is engineered to form covalent bonds with ligands that have different functional reporters.
(0003925) |
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Products: HaloLink™ Magnetic Beads | HaloLink™ Resin | HaloTag® Alexa Fluor® 488 Ligand | HaloTag® Amine (O2) Ligand | HaloTag® Amine (O4) Ligand | HaloTag® Biotin Ligand | HaloTag® Coumarin Ligand | HaloTag® diAcFAM Ligand | HaloTag® Iodoacetamide (O2) Ligand | HaloTag® Iodoacetamide (O4) Ligand | HaloTag® Oregon Green® Ligand | HaloTag® PEG-Biotin Ligand | HaloTag® pHT2 Vector | HaloTag® Succinimidyl Ester (O2) Ligand | HaloTag® Succinimidyl Ester (O4) Ligand | HaloTag® Thiol (O4) Ligand | HaloTag® TMR Ligand |
| 21. |
Heikkinen, L.S., Kazlauskas, A., Melén, K., Wagner, R., Ziegler, T., Julkunen, I. and Saksela, K.
(2008)
Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling.
J. Biol. Chem.
283
,
5719–27
.
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| |
Notes:
The authors identified the N-terminal Src homology 3 (SH3) domain-binding motif of Crk and CrkL as the preferred binding partner of nonstructural protein 1 (NS1), an important virulence factor of the influenza A virus. Interaction of NS1 with Crk, CrkL and other SH3 domain-containing proteins p85, p85β and Eps8L1 was investigated by protein pull-down assays. Expression constructs for biotinylated Crk, CrkL, p85, p85β and Eps8L1 were created by amplifying the 123 amino acid biotin acceptor domain from Propionibacterium shermanii from the PinPoint Xa-T Vector and inserting it upstream of the protein-coding sequences in a pGEX vector derivative. These constructs and a construct encoding Myc-tagged NS1 were transfecting into 293FT cells, and the biotinylated proteins were immobilized from the cell lysate using TetraLink™ Tetrameric Avidin Resin. Any Myc-NS1 that bound to the immobilized protein was detected using Western Blot analysis and an anti-Myc antibody. The authors also investigated the ability of wildtype NS1 or NS1 mutants to inhibit interferon-induced gene expression. A reporter plasmid was created by cloning an interferon-stimulated response element upstream of a minimal thymidine kinase promoter driving firefly luciferase expression. A vector containing Renilla luciferase was used as a control to normalize transfection efficiency. Huh-7 cells were cotransfected with the firefly and Renilla luciferase reporter constructs (0.2µg and 5ng, respectively), treated with interferon-β and lysed using the Passive Lysis Buffer. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System.
(0003803) |
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Products: Dual-Luciferase® Reporter Assay System | TetraLink™ Tetrameric Avidin Resin |
| 22. |
Birdsey GM, Dryden NH, Amsellem V, Gebhardt F, Sahnan K, Haskard DO, Dejana E, Mason JC, Randi AM.
(2008)
Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin.
Blood
111
,
33498-33506
.
|
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Notes:
These authors showed that the ETS transcription factor Erg interacts with the VE-cadherin promoter region and regulates endothelial apoptosis through this interaction. They demonstrated that inhibition of Erg by siRNA resulted in decreased VE-cadherin mRNA and protein levels, and showed that Erg interacts with the VE-cadherin promoter using a CHIP assay. To show the functional relevance of this interaction, HeLa cells were transfected with a pGL4 Vector containing the VE-cadherin promoter region and an expression vector containing Erg2 cDNA. In this reporter assay, Erg overexpression resulted in ~1.8 fold transactivation of VE-cadherin promoter activity, as measured using the Dual-Luciferase® Reporter Assay System. Inhibition of Erg in human umbilical vein endothelial cells also resulted in a loss of viability and an increase in activation of caspase 3 and caspase 7. The authors showed that apoptosis could be significantly decreased by overexpression of VE-cadherin, indicating that Erg regulates survival partially via its interaction with VE-cadherin. The Caspase-Glo® 3/7 Assay was used to measure caspase activity in these experiments.
(0003872) |
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Products: Caspase-Glo® 3/7 Assay | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 23. |
Cavallaro, M., Mariani, J., Lancini, C., Latorre, E., Caccia, R., Gullo, F., Valotta, M., DeBiasi, S., Spinardi, L., Ronchi, A., Wanke, E., Brunelli, S., Favaro, r., Ottolenghi, S. and Nicolis, S.K.
(2008)
Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants
Development
135
,
541-557
.
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Notes:
The authors of this paper investigated the role of Sox2 in neuronal differentiation. Neurospheres were derived from the brains of normal and Sox2 hyphomorphic mice and used to generate differentiated neural cells. In astroglia from cultures containing a Sox2-GFP-expressing lentivirus, ectopic expression of Sox2 correlated with reduced GFAP expression. The authors investigated the role of Sox2 by amplifying binding sites upstream of the GFAP promoter and cloning them upstream of the thymidine kinase promoter in the pRL-TK vector. The Dual-Luciferase Assay System was used to analyze the effect of Sox2 expression on the luciferase reporter gene. The DeadEnd™ Fluorometric TUNEL System was also used to assess apoptosis in some of the neurosphere-derived cultures.
(0003953) |
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Products: DeadEnd™ Fluorometric TUNEL System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | pRL-TK Vector |
| 24. |
Tao, R.H. and Maruyama, I.N.
(2008)
All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells.
J. Cell Sci.
121
,
3207–3217
.
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Notes:
The CheckMate™ Mammalian Two-Hybrid System was used to explore the dimerization of epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases without ligand. The intracellular domains of EGFR, ErbB2, ErbB3 and ErbB4 were amplified and cloned into both the pACT and pBIND Vectors. Transfection into NIH3T3 cells in 12-well plates occurred with 0.3 μg of pG5luc Vector (the reporter vector), 0.2 μg of pACT Vector or an equimolar amount of pACT construct, and 0.1 μg of pBIND Vector or an equimolar amount of pBIND construct. After 24 hours, the cells were lysed and luciferase activity assessed using the Dual-Luciferase® Reporter Assay System.
(0003993) |
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Products: CheckMate™ Mammalian Two-Hybrid System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 25. |
Sueyoshi, T., Moore, R., Sugatani, J., Matsumura, Y. and Negishi, M.
(2008)
PPP1R16A, the membrane subunit of protein phosphatase 1{beta}, signals nuclear translocation of the nuclear receptor CAR.
Mol. Pharmacol.
73
,
1113-1121
.
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Notes:
This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT® Coupled Reticulocyte Lysate System and labeled with 35S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase® Reporter Assay System.
(0003752) |
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Products: CheckMate™ Mammalian Two-Hybrid System | Dual-Luciferase® Reporter Assay System | Sequencing Grade Modified Trypsin | TNT® SP6 Coupled Reticulocyte Lysate System | TNT® T3 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System |