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Neuropsychopharmacology Feb. 11, (epub ahead of print). Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference. 2009

Brunzell, D.H., Mineur, Y.S, Neve, R.L. and Picciotto, M.R.

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. (3956)

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Vet. Rec. 164, 44–47. Prevalence of thermophilic Campylobacter species in household cats and dogs in Ireland. 2009

Acke, E., McGill, K., Golden, O., Jones, B.R., Fanning, S. and Whyte, P.

Notes: To examine the prevalence of Campylobacter species in asymtomatic carriers that can pass the bacteria onto humans, rectal swabs were collected from 147 dogs and 35 cats in Ireland and cultured on various diagnostic plates. The Wizard® Genomic DNA Purification System was used to isolate DNA from any suspect Campylobacter cultures. The purified DNA was used in multiplex PCR and RFLP to determine which species of Campylobacter was present. (4016)

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Nucl. Acids Res. 37, 5343–5352. Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance. 2009

Oberto, J., Breuil, N., Hecker, A., Farina, F., Brochier-Armanet, C., Culetto, E. and Forterre, P.

Notes: In this paper, the role of the KAE1/osgep/ygjD gene family, a universally conserved gene set without an assigned function, was investigated in yeast and Caenorhabditis elegans. Genomic DNA was isolated from Saccharomyces cerevisiae using the Wizard® SV Genomic DNA Purification System. This purified DNA was then used in PCR. (4065)

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Drug Metab. Dispos. 37, 1759–1768. Quantitative analysis of UDP-glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers. 2009

Izukawa, T., Nakajima, M., Fujiwara, R., Yamanaka, H., Fukami, T., Takamiya, M., Aoki, Y., Ikushiro, S., Sakaki, T. and Yokoi, T.

Notes: This study examined the expression levels of each UGT isoform in human liver and evaluated the variability between individuals. Total RNA from appropriate human tissues or various cell lines was used for RT-PCR of various human UDP-glucuronosyltransferases (UGT) cDNAs. The amplimers were cloned into the pTARGET™ Mammalian Expression Vector and verified by sequencing. The UGT vectors were linearized by restriction enzyme digestion and used for standards in real-time RT-PCR analysis. (4034)

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Nucl. Acids Res. 37, 78–95. Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage. 2009

Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.

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. (4031)

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Infect. Immun. 77, 3234–43. Sab, a novel autotransporter of locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21, contributes to adherence and biofilm formation. 2009

Herold, S., Paton, J.C. and Paton, A.W.

Notes: To try to understand the mechanism by which certains strains of Shiga-toxigenic E.coli adhere to host intestinal epithelium, the authors characterized the novel autotransporter protein Sab. Expression levels and protein localization were examined by Western blot analysis and enzyme-linked immunosorbent assay. The mouse anti-Sab antibody used in these studies was raised against an N-terminal His6-Sab fusion protein purified using the HisLink™ Resin. (4102)

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Appl. Environ. Microbiol. 75, 5600–6. Single nucleotide polymorphism-based diagnostic system for crop-associated Sclerotinia species. 2009

Andrew, M. and Kohn, L.M.

Notes: The authors developed a single nucleotide polymorphism (SNP)-based assay to distinguish four Sclerotinia species. The assay consisted of amplification of a 300bp intergenic spacer and portions of the calmodulin and ras genes, followed by Southern blot using species-specific, radiolabeled probes. Amplifications were performed using the GoTaq® Colorless Master Mix, 0.2µM of each primer and 10–20ng of template DNA. (4098)

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Clin. Can. Res. 15, 7562–70. Smoking-related gene expression in laser capture-microdissected human lung. 2009

Tan, X.L., Wang, T., Xiong, S., Kumar, S.V., Han, W. and Spivack, S.D.

Notes: The authors characterized differential expression of several carcinogen metabolism genes in human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissues in smokers, former smokers and people who have never smoked. They combined laser capture microdissection (LCM) and quantitative RT-PCR. RNA was isolated from paired microdissected malignant and nonmalignant lung tissue, 100ng of total RNA was reverse transcribed in a 20µl reaction, then 1µl of cDNA was amplified by real-time PCR using an ABI PRISM® 7500HT sequence detection system, GoTaq® Flexi DNA Polymerase and gene-specific primers. The expression level for each gene was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that expression of cytochrome P450 1B1 and glutathione-S-transferase P1 in AC, but not BEC, tissue was strongly associated with exposure to tobacco. (4095)

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J. Biol. Chem. 284, 19402–11. Structural Determinants of G-protein α Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2) 2009

Kimple, A.J., Soundararajan, M., Hutsell, S.Q., Roos, A.K., Urban, D.J., Setola, V., Temple, B.R.S., Roth, B.L., Knapp, S.K., Willard, F.S. and Siderovsk, D.P.

Notes: The authors created a triple mutant of Regulator of G-Protein Signaling Protein 2 (RGS2) to characterize the structural features responsible for its selectivity in binding to the Gαq or Gαi/o subunits of GTPase-accelerating protein (GAP). The RGS2 enhances the termination of G-protein coupled signaling by enhancing GAP. RGS proteins are considered key modulators of G Protein-Coupled Receptor (GPCR) signaling based on their ability to accelerate GTP hydrolysis. The GloSensor™ cAMP assay was used to assess the level of GPCR activity and indicate which structural determinants of RGS2 affect binding to Gα subunits of GAP.

HEK293T cells were transiently co-transfected with expression vectors for the GloSensor™ cAMP biosensor and the Gi-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Treatment of transfected cells with forskolin produced an increase in luminescence from the cAMP sensor, reflecting direct activation of adenylyl cyclase by forskolin. Quinpirole, a dopamine D2 receptor agonist, produced a dose-dependent inhibition of cAMP production. Inhibition of forskolin-stimulated cAMP production was assessed after activation of the D2 receptor with various concentrations of quinpirole to compare IC50 values for the empty vector, wild type RGS2 and triple mutant RGS2. Cellular expression of the triple mutant resulted in a significantly higher IC50 for quinpirole (762nM versus 18 nM for empty vector), indicating that the three point mutations weaken Gαi subunit binding responsible for enhanced GTPase activity.

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Plant Physiol. 150, 1356–1367. Sucrose control of translation mediated by an upstream open reading frame-encoded peptide. 2009

Rahmani, F., Hummel, M., Schuurmans, J., Wiese-Klinkenberg, A., Smeekens, S. and Hanson, J.

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. (4023)

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Ann. Oncol. 20, 879-884. The importance of KRAS mutations and EGF61A>G polymorphism to the effect of cetuximab and irinotecan in metastatic colorectal cancer. 2009

Garm Spindler, K.L., Pallisgaard ,N., Rasmussen, A.A., Lindebjerg, J., Andersen, R.F., Crüger, D., and Jakobsen, A.

Notes: These authors used the Maxwell® 16 System to isolate genomic DNA from whole blood and normal colonic tissue samples. The DNA was used in genotype analysis, testing for wildtype and mutant KRAS genes, and for various EGFR-related polymorphisms. The results were used in a research study testing the relationship between various genotypes and response to different treatment regimens. (3961)

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J. Bacteriol. 190, 1912–21. Borrelia burgdorferi uniquely regulates its motility genes and has an intricate flagellar hook-basal body structure. 2008

Sal, M.S., Li, C., Motalab, M.A., Shibata, S., Aizawa, S. and Charon, N.W.

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. (3885)

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Malaria Journal Oct 29:7, 223. A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking. 2008

Daniels R, Volkman SK, Milner DA, Mahesh N, Neafsey DE, Park DJ, Rosen D, Angelino E, Sabeti PC, Wirth DF, Wiegand RC.

Notes: These authors used the Maxwell® 16 System to isolate DNA from frozen whole blood samples infected with Plasmodium falciparum. The isolated DNA was used in a qPCR-based SNP genotyping assay that sought to uniquely identify the parasites based on their SNP marker profile. (3962)

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Cancer Res. 68, 5639–5647. A special linker between macrophage and hematopoietic malignant cells: membrane form of macrophage colony-stimulating factor. 2008

Wang, L., Zheng, G.G., Ma, C.H., Lin, Y.M., Zhang, H.Y., Ma, Y.Y., Chong, J.H. and Wu, K.F.

Notes: To examine the role of the membrane form of macrophage colony–stimulating factor(mM-CSF) in the hematopoietic system, RT-PCR was used amplify the cDNA of human mM-CSF from J6-1 cells, a human leukemia cell line. The PCR product was digested and cloned into the pTargeT™ Mammalian Expression Vector. After sequencing to verify the sequence, the construct and empty pTargeT™ Mammalian Expression Vector were purified and used to transfect Namalwa and Ramos cells, human Burkitt’s lymphoma cell lines, in 24-well plates. The transfected cells were then selected for stable expression of the transfected vector using 1.4 mg/ml G418. Expression of mM-CSF and neomycin (in the empty vector) was confirmed using RT-PCR. These cells were injected into mice and the oncogenicity of the cells determined using antibody staining of tissues. (3989)

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Proc. Natl. Acad. Sci. USA 105, 8914-8919. An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site. 2008

Abdel-Latief, M., Garbe, L.A., Koch, M., and Ruther, J.

Notes: These authors amplified and characterized a putative epoxide hydrolase gene from the jewel wasp Nasonia vitripennis. PCR fragments were amplified from genomic DNA, purified from gels using the Wizard® SV Gel and PCR Clean Up System and then subcloned into the pGEM®-T Easy Vector. The plasmid DNA was purified using the PureYield™ Midiprep System. Linearized plasmids were used for in vitro transcription of RNA for use in RNA interference experiments. (3903)

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J. Biol. Chem. 283, 21579–87. ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action. 2008

Betzenhauser, M.J., Wagner, L.E. 2nd, Iwai, M., Michikawa, T., Mikoshiba, K. and Yule, D.I.

Notes: The authors examined the different ATP sensitivities of inositol (1,4,5)-triphosphate receptor (InsP3R) isoforms InsP3R1, InsP3R2 and InsP3R3. To compare the ATP-binding properties of InsP3R2 and InsP3R3, nucleotide sequences encompassing the ATP-binding domains were amplified by PCR and cloned into the pFN2A (GST) Flexi® Vector. The ATP-binding sites were expressed as glutathione-S-transferase (GST) fusion proteins in BL21(DE3)pLysS cells. Fusion proteins were purified, and the GST tag removed by cleavage with tobacco etch virus (TEV) protease. Purified proteins were then used in a fluorescent ATP-binding assay. (3901)

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Biol. Reprod. 79, 594–7. Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially? 2008

Bermejo-Alvarez, P., Rizos, D., Rath, D., Lonergan, P. and Gutiérrez-Adán, A.

Notes: To determine whether oocytes are able to select X-bearing or Y-bearing spermatozoa, the authors performed in vitro fertilization of bovine oocytes with X-sorted semen, Y-sorted semen, a mixture of X- and Y-sorted semen, and unsorted semen. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome-specific target for gender assignment and a bovine-specific satellite sequence as a control. PCRs were performed using GoTaq® Flexi DNA Polymerase (1 unit per 25µl reaction), and amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. (3881)

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Appl. Environ. Microbiol. 74, 312-318. Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitrosococcus oceani ATCC 19707. 2008

El Sheikh, A.F., Poret-Peterson, A.T. and Klotz, M.G.

Notes: These authors investigated the amo operon of the marine ammonia oxidizer Nitrosococcus oceani. The bacteria were grown at 30°C for 3 weeks in 200-400ml batch cultures in artificial seawater in the dark without shaking. Genomic DNA was isolated from cells in stationary phase using the Wizard® Genomic DNA Purification Kit. The isolated DNA was then used for PCR analysis. (3740)

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J. Biol. Chem. 283, 8218–28. Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway. 2008

Miao, Q., Sun, Y., Wei, T., Zhao, X., Zhao, K., Yan, L., Zhang, X., Shu, H. and Yang, F.

Notes: The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT® Coupled Transcription Translation System and [35S] methionine. (3889)

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Genetics 178, 1415–29. Comparative genetics of sex determination: masculinizing mutations in Caenorhabditis briggsae. 2008

Kelleher, D.F., de Carvalho, C.E., Doty, A.V., Layton, M., Cheng, A.T., Mathies, L.D., Pilgrim, D. and Haag, E.S.

Notes: The authors characterized masculinizing mutations of the female-promoting tra genes in Caenorhabditis briggsae (Cb-tra). Using RT-PCR, the authors monitored the levels of full-length Cb-tra mRNA and a novel splice variant; actin mRNA was amplified as a control. RT-PCR was carried out using the AccessQuick™ RT-PCR System and RNA from 5–10 worms per 50µl reaction. (3892)

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Mol. Cancer Res. 6, 546–54. CXCL16 functions as a novel chemotactic factor for prostate cancer cells in vitro. 2008

Lu, Y., Wang, J., Xu, Y., Koch, A.E., Cai, Z., Chen, X., Galson, D.L., Taichman, R.S. and Zhang, J.

Notes: The authors sought to detect expression of the chemokine CXCL16 and its receptor, CXCR6, in prostate cancer cell lines and in benign and malignant prostate cancer tissues. The Access RT-PCR System was used to amplify and detect CXCL16 and CXCR6 mRNA in these cells and tissues. Each RT-PCR contained 1µg of total RNA, and amplifications were carried out for 35 cycles. Amplified products were detected on a 1.5% ethidium bromide-stained agarose gel. (3836)

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Plant Physiol. 146, 1469–81. Deregulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant. 2008

Covshoff, S., Majeran, W., Liu, P., Kolkman, J.M., van Wijk, K.J. and Brutnell, T.P.

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. (3883)

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Forensic Sci. Int. Genet. 3, 14–21. Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA. 2008

Krenke, B.E., Nassif, N., Sprecher, C.J., Knox, C., Schwandt, M. and Storts, D.R.

Notes: The authors describe the developmental validation of the Plexor® HY System, a quantitative PCR assay that simultaneously quantifies total human and male DNA. Validation studies examined: (1) human specificity, (2) sensitivity, (3) quantitation of degraded DNA, (4) impact of inhibitors, (5) male/female mixture and Y-assay male specificity, (6) reproducibility and concordance and (7) population studies. (3969)

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Stem Cells 26, 1841-1849. Embryonic Stem Cells as a Platform for Analyzing Neural Gene Transcription 2008

Zhang, X., Horrell, S.A., Delaney, D., Gottlieb, D.I.

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 controlling 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. (3918)

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DNA Research 15, 137-149. Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products. 2008

Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.

Notes: These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

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