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| 1. |
Yu, M.J., Miller, R.L., Uawithya, P., Rinschen, M.M., Khositseth, S., Braucht, D.W., Chou, C.L., Pisitkun, T., Nelson, R.D. and Knepper, M.A.
(2009)
Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct.
Proc. Natl. Acad. Sci. U S A
106
,
2441–2446
.
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Notes:
The authors used a systems biology approach to examine the transcriptional regulation of water channel aquaporin-2 (AQP2). A 1,511bp fragment from the 5´-flanking region of the mouse AQP2 gene was amplified from mouse tail DNA and cloned into the pGEM®-T Vector. This construct was then digested with two restriction enzymes and cloned into a double-digested pGL3-Basic Vector. Full length Elf3, Elf5 and Ehf cDNA, members of the ETS family of transcriptional regulators, were amplified, sequenced and ligated into the pTARGET™ Mammalian Expression Vector. LLCPK1 cells were cotransfected with AQP2-pGL3 reporter and one of the pTARGET™ constructs. Reporter activity was measured using 20µl of cell lysate in a luciferase assay.
(0004033) |
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Products: pGEM®-T Vector | pGEM®-T Vector System I | pGEM®-T Vector System II | pGL3 Basic Vector | pTARGET™ Mammalian Expression Vector System | pTARGET™ Vector |
| 2. |
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 |
| 3. |
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 |
| 4. |
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 |
| 5. |
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 |
| 6. |
Wruck, C.J., Götz, M.E., Herdegen, T., Varoga, D., Brandenburg, L-O. and Pufe, T.
(2008)
Kavalactones protect neural cells against amyloid β peptide-induced neurotoxicity via ERK1/2-dependent Nrf2-activation
Molecular Pharmacology Fast Forward
March 11, 2008
,
epub ahead of print
.
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Notes:
The accumulation of the toxic form of the Amyloid-β peptide is known to induce oxidative damage in the brain. Although treatment with antioxidants has not proven effective at controlling AD symptoms, inducing the natural systems in the brain that protect from oxidative damage may provide a possible therapeutic approach. A host of antioxidant and detoxifying enzymes are upregulated by binding of the Nrf2 transcription factor to the ARE (antioxidant response element) regulatory sequence. The authors used a Dual Luciferase® Reporter Assay to assess modulation of gene activity through ARE by kavalactones. Kavalactones are compounds found in the roots and rhizomes of Kava (Piper methysticum), a plant cultivated an used in some Pacific societies for medicinal and social uses. The ARE1 region from the rat NAD(P)H:quinone oxidoreductase-1 gene was placed upstream of a pGL3 firefly luciferase reporter construct and cotransfected along with a pRL-TK Renilla control construct into PC12 or C6 cells. The data show induction of luciferase activity by kavalactones. Further investigation shows that the kavalactones promote Nrf2 stabilization possibly through the ERK1/2 pathway.
(0003859) |
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Products: Dual-Luciferase® Reporter Assay System | pGL3-Basic Vector | pGL3-Control Vector | pGL3-Enhancer Vector | pGL3-Promoter Vector | pRL-TK Vector |
| 7. |
Sal, M.S., Li, C., Motalab, M.A., Shibata, S., Aizawa, S. and Charon, N.W.
(2008)
Borrelia burgdorferi uniquely regulates its motility genes and has an intricate flagellar hook-basal body structure.
J. Bacteriol.
190
,
1912–21
.
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Notes:
The authors investigated gene transcription within periplasmic flagella of Borrelia burgdorferi, which are composed of a basal body, hook and filament, to determine if hook formation influences flagellin gene expression. They used insertion mutagenesis to construct strains with mutated versions of the hook structural gene flgE that were disrupted by a kanamycin-resistance cassette. The flgE gene and antibiotic-resistance cassette were amplified by PCR and cloned into the pGEM®-T Vector. To assess the effect of flgE disruption on the transcription of filament proteins FlaA and FlaB, quantitative RT-PCR was performed; enolase was used as an internal control. Negative controls without the reverse transcriptase were included for each sample.
(0003885) |
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Products: pGEM®-T Easy Vector System I | Reverse Transcription System |
| 8. |
Scorpio, D.G., Leutenegger, C., Berger, J., Barat, N., Madigan, J.E. and Dumler, J.S.
(2008)
Sequential analysis of Anaplasma phagocytophilum msp2 transcription in murine and equine models of human.
Clin. Vaccine Immunol.
15
,
418–424
.
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Notes:
The authors examined the pattern of Anaplasma phagocytophilum msp2 expression, a gene that modulates with little immune pressure and has decreased virulence with prolonged in vitro passage. C57BL/6J mice were inoculated with HL-60 cells infected with low-passage (passage 5) or high-passage (passage 26). Blood samples were taken 2–21 days post-inoculation, and total RNA was isolated. The purified RNA was subjected to RT-PCR, cloned into the pGEM®-T Easy Vector, transformed and plated. Plasmids were purified using the Wizard® SV 96 Plasmid DNA Purification System, and the insert size analyzed after EcoRI digestion. The inserts were sequenced, aligned with A. phagocytophilum Webster strain msp2 references using ClustalX and the diversity of msp2 transcripts divided into low- or high-passage bacteria.
(0003975) |
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Products: pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | Wizard® SV 96 Plasmid DNA Purification System |
| 9. |
Lee, B.C., Le, D.T. and Gladyshev, V.N.
(2008)
Mammals reduce methionine-S-sulfoxide with MsrA and are unable to reduce methionine-R-sulfoxide, and this function can be restored with a yeast reductase.
J. Biol. Chem.
283
,
28361–28369
.
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Notes:
The C-terminus of free methionine-R-sulfoxide reductase (fRMsr) gene, a yeast gene that can generate methionine from methionine-S-sulfoxide and methionine-R-sulfoxide, was tagged with six histidines before being cloned into the pCI-neo Mammalian Expression Vector. This construct was transfected into SK-Hep1 hepatocytes with or without the Clonegene pEGFP-N1 Vector. Using 800 μg/ml G418 sulfate, a stable cell line was selected. This cell line was then tested for response to oxidative stress.
(0003985) |
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Products: pCI-neo Mammalian Expression Vector |
| 10. |
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 |
| 11. |
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 |
| 12. |
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
.
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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 |
| 13. |
Covshoff, S., Majeran, W., Liu, P., Kolkman, J.M., van Wijk, K.J. and Brutnell, T.P.
(2008)
Deregulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant.
Plant Phys.
146
,
1469–81
.
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Notes:
The authors identified the maize homolog of hcf136 (Zmhcf136), a gene involved in photosynthesis, and used an RNA blot to determine if ZmHcf136 transcripts accumulate preferentially in mesophyll cells. DNA probes for Zmhcf136 and several cell-specific markers were generated by PCR using GoTaq® Green Master Mix, gel purified and radiolabeled prior to use in the RNA blots. To examine differences in protein accumulation and localization in wildtype and hcf136 mutants, proteins from subcellular fractions were subjected to two-dimensional gel electrophoresis, and spots of interest were excised, digested with Sequencing Grade Modified Trypsin, then analyzed by electrospray ionization-tandem mass spectrometry.
(0003883) |
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Products: GoTaq® Green Master Mix | Sequencing Grade Modified Trypsin |
| 14. |
Buhr, F., El Bakkouri, M., Valdez, O., Pollmann, S., Lebedev, N., Reinbothe, S. and Reinbothe, C.
(2008)
Photoprotective role of NADPH:protochlorophyllide oxidoreductase A.
Proc. Natl. Acad. Sci. U S A
105
,
12629–12634
.
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Notes:
To examine the role of NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) during greening, the PORA gene was mutated by changing each of the four conserved Cys to Ala using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting genes were subcloned into an expression vector and transformed into XL1-Blue cells. The proteins were expressed either in E. coli cells after IPTG induction or in a wheat germ extract system and tested for interaction with Pchlide molecules and NADPH.
(0003994) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 15. |
Cameron, A.D., Volar, M., Bannister, L.A. and Redfield, R.J.
(2008)
RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenzae.
Nucleic Acids Res.
36
,
10–20
.
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Notes:
The authors examined the role of sxy expression in hypercompetence of Haemophilus influenza. The 1.8kb sxy gene was subcloned into the pALTER®-1 Vector and point mutations made using the Altered Sites® II in vitro Mutagenesis System. The mutated gene was sequenced and subcloned back into the original plasmid. The wildtype and mutant sxy genes were transcribed, dephosphorylated and labeled with 32P ATP. The end-labeled RNAs were then subjected to S1 nuclease mapping. The E. coli S30 Extract System for Linear Templates was used with the wildtype and mutant sxy constructs and the expression levels of the expressed protein measured by spot blotting.
(0003995) |
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Products: E. coli S30 Extract System for Linear Templates | Altered Sites® II in vitro Mutagenesis System |
| 16. |
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 |
| 17. |
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 |
| 18. |
Hahn, C.K., Ross, K.N., Warrington, I.M., Mazitschek, R., Kanegai, C.M., Wright, R.D., Kung, A.L., Golub, T.R. and Stegmaier, K.
(2008)
Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation.
Proc. Natl. Acad. Sci. USA
105
,
9751-9756
.
|
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Notes:
The authors designed a high-throughput gene-expression screen to identify compounds that induce a neuroblastoma gene signature in BE(2)-C cells. The screen used Valproic Acid (VPA), an inhibitor of histone deacetylase, as an enhancer. The top hit from the screen was all-trans retinoic acid (ATRA). The authors proposed that ATRA + VPA would reduce cell viability and might induce apoptosis by inducing genes associated with terminal differentiation. They used the CellTiter-Glo® Luminescent Cell Viability Assay in a 96-well format to assess the effects of ATRA and a variety of inhibitors of histone deacetylase on BE(2)-C cell viability.
(0003931) |
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Products: CellTiter-Glo® Luminescent Cell Viability Assay |
| 19. |
Simonetti, M., Giniatullin, R., and Fabbretti, E.
(2008)
Mechanism mediating the enhanced transcription of P2X3 receptor gene by calcitonin gene related peptide in trigeminal sensory neurons.
J. Biol. Chem.
May 6
,
Epub (ahead of print)
.
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Notes:
These authors investigated the mechanism of action of the migraine mediator calcitonin gene-related peptide (CGRP), which is known to sensitize the P2X3 pain receptors to increase impulse flow to brain stem trigeminal nuclei. They showed that CAM KII inhibitors prevented CGRP-induced upregulation of P2X3 mRNA using real-time RT-PCR, and then confirmed that active CAM KII was involved in the signaling mechanism by staining with Anti-ACTIVE® CAM KII Antibodies. The immunoreactivity was upregulated by CGRP treatment of trigeminal neurons, and the distribution of the active CAM KII was localized to the membrane region. They then examined the mechanism of transcriptional activation of the P2X3 pain receptor genes and showed that the transcription factor CREB, which is known to be dependent on CAM KII for activation and nuclear localization, was involved. The authors also investigated the role of BDNF in the process, because CGRP is known to promote BDNF expression by trigeminal neurons, and because BDNF is thought to be involved in pain processing. They used the BDNF Emax Immunoassay System to measure BDNF levels in culture media after application of CGRP to neuronal cell cultures, and demonstrated that there was a fourfold increase in BDNF after CGRP exposure. They also showed that BDNF promoted CAMKII and CREB activation.
(0003873) |
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Products: Anti-ACTIVE® CaM KII pAb, Rabbit, (pT286) | BDNF Emax® ImmunoAssay System |
| 20. |
Nanashima, N., Akita, M., Yamada, T., Shimizu, T., Nakano, H., Fan, Y. and Tsuchida, S.
(2008)
The hairless phenotype of the Hirosaki hairless rat is due to the deletion of an 80-kb genomic DNA containing five basic keratin genes.
J. Biol. Chem.
283
,
16868–75
.
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| |
Notes:
The mutation responsible for the hairless phenotype was linked to a 80kb deletion of chromosome 7q36. Because many basic keratin genes are located at 7q36, the authors examined keratin gene expression in the Hirosaki hairless rat using RT-PCR. Expression of kb21, kb23 and kb26 was not detected, whereas other basic keratin genes were expressed. RT-PCR was performed using the AccessQuick™ RT-PCR System and 0.5µg of total RNA isolated from rat skin for 21–30 cycles.
(0003887) |
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Products: AccessQuick™ RT-PCR System |
| 21. |
Chang, Q,. Bhatia, D., Zhang, Y., Meighan, T., Castranova, V., Shi, X. and Chen, F.
(2008)
Incorporation of an internal ribosome entry site-dependent mechanism in arsenic-induced GADD45 alpha expression.
Cancer Res.
2007
,
6146–54
.
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| |
Notes:
Trivalent arsenic (As3+) induces expression of growth arrest- and DNA damage-induced gene 45α (GADD45α), which interacts with intracellular signaling molecules involved in cell cycle regulation, apoptosis and immune response. To characterize GADD45α mRNA expression patterns, total RNA was isolated from untreated (growing) and As3+-treated (arrested) BEAS-2B cells, and GADD45α mRNA was amplified using the AccessQuick™ RT-PCR System. GADD45α mRNA levels were undetectable in growing cells but increased in a time-dependent manner in arrested cells. Reverse transcription was carried out at 45°C for 50 minutes, followed by 35 cycles of PCR.
(0003766) |
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Products: AccessQuick™ RT-PCR System |
| 22. |
Zhang, X., Horrell, S.A., Delaney, D., Gottlieb, D.I.
(2008)
Embryonic Stem Cells as a Platform for Analyzing Neural Gene Transcription
Stem Cells
26
,
1841-1849
.
|
| |
Notes:
The authors note that while spatially and temporally specific gene transcription is a fundamental process in the normal development of mammalian stem cells, transcription in stem cells is currently studied by a set of methodologies with significant limitations. For instance, transient transfections analyze gene regulatory elements in nonchromosomal context. Using transgenic mice places transgenes in chromosomal context, however the chromosomal site where the transgene is inserted strongly influences the transgenes expression. As well, the need to make transgenic mice limits the number of experiments that can be done. ESCs can overcome these limitation. Undifferentiated stem cells are suitable for genetic engineering approaches such as gene targeting and recombinase-mediated cassette exchanges. By using such techniques, precisely planned alteration of native genes such as insertion of reporters, deletions of nearby or distant DNA sequences and mutational substitutions can be made. The authors wanted to analyze the Olig2 gene, a helix-loop-helix transcription factor expressed in the developing nervous system. Because Olig2 plays a central role in differentiation, understanding how it is regulated is important to understanding the larger transcriptional network controling development.
To this end, the authors used vectors for transient transfection experiments, constructed by amplifying regions of the Olig2 gene by PCR using primers tailed with appropriate restrictions sites and cloning the fragments into a pGL3 Luciferase Reporter Vector (Cat. # E1741, e1751, e1761, e1771). Promoter-reporter DNA was transfected into ESCs, cells were cultured 24 hours, then luciferase assays (Promega, type not specified) used to measure transgene expression.
(0003918) |
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| 23. |
Gierman, H.J., Indemans, M.H., Koster, J., Goetze, S., Seppen, J., Geerts, D., van Driel, R. and Versteeg, R.
(2007)
Domain-wide regulation of gene expression in the human genome.
Genome Res.
17
,
1286-1295
.
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Notes:
The authors explored the possibility of a domain-based level of gene expression regulation for chromosomes by integrating the green fluorescent protein (GFP) reporter in 90 different locations in cultured human cells. This integration was accomplished by infecting human embryonic kidney cells (HEK293) with a lentiviral construct carrying the GFP gene under the control of the ubiquitously expressed human phosphoglycerate kinase (PGK), sorting GFP-positive cells by FACS and selecting clones for expansion. Genomic DNA was isolated from the various clones using the Wizard® SV Genomic DNA Purification System and analyzed by PCR or restriction digested for Southern blotting.
(0003743) |
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Products: Wizard® SV Genomic DNA Purification System |
| 24. |
Gluckman PD, Lillycrop KA, Vickers MH, Pleasants AB, Phillips ES, Beedle AS, Burdge GC, Hanson MA.
(2007)
Metabolic plasticity during mammalian development is directionally dependent on early nutritional status.
Proc. Natl. Acad. Sci. U S A
104
,
12796-12800
.
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Notes:
To examine how exposure to leptin in utero affects hepatic gene expression and epigenetic status in adulthood, pregnant Wistar rats were fed a standard diet or 30% undernutrition. The three-day-old pups were exposed to saline or leptin for 10 days, weaned and either fed a standard or high-fat diet. On day 170, the rats were sacrificed and tissues snap frozen. Five micrograms of genomic DNA was isolated from rat liver using the Wizard® SV Genomic DNA Purification System. Then 25ng of the purified DNA was digested with methylation-sensitive restriction enzymes, and the glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor alpha (PPARα) fragments were amplified by real-time PCR.
(0003744) |
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Products: Wizard® SV Genomic DNA Purification System |
| 25. |
Bodó, E., Tobin, D.J., Kamenisch, Y., Bíró, T., Berneburg, M., Funk, W. and Paus, R.
(2007)
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
Am. J. Pathol.
171
,
1153-1167
.
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Notes:
To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan® Assay.
(0003746) |
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Products: AMV Reverse Transcriptase | Random Primers | Wizard® SV Genomic DNA Purification System |