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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. |
Sampathi, S., Bhusari, A., Shen, B. and Chai, W.
(2009)
Human flap endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance.
J. Biol. Chem.
284
,
3682–3690
.
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Notes:
The authors explored the role of a DNA replication factor, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. PCR was used to add a myc tag to the N terminus of FEN1 cDNA. The amplimer was gel purified, digested with NheI and SmaI, and cloned into the same sites in the pCI-neo Mammalian Expression Vector. The insert was confirmed by sequencing.
(0004030) |
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Products: pCI-neo Mammalian Expression Vector |
| 4. |
Yamada, K., Takahashi, M., Hoshino, Y., Takahashi, H., Ichiyama, K., Tanaka, T. and Okamoto, H.
(2009)
Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells.
J. Gen. Virol.
90
,
457–462
.
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Notes:
The authors created a full-length infectious cDNA clone of a genotype 3 hepatitis E virus (strain JE03-1760F) for use in cell culture. The full-length ORF2 sequence of the JE03-1760F genome was amplified and cloned into the pCI Mammalian Expression Vector. The construct was transfected into PLC/PRF/5 cells for 3 days then analyzed by Western blotting.
(0004029) |
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Products: pCI Mammalian Expression Vector |
| 5. |
Staniszewska, I., Sariyer, I.K., Lecht, S., Brown, M.C., Walsh, E.M., Tuszynski, G.P., Safak, M., Lazarovici, P. and Marcinkiewicz, C.
(2008)
Integrin alpha9 beta1 is a receptor for nerve growth factor and other neurotrophins.
J. Cell Sci.
121
,
504–13
.
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Notes:
The authors investigated the ability of α9β1 integrin to act as a neurotrophin receptor and affect cell signaling pathways. As part of the study, RT-PCR was used to detect the presence of other neurotrophin receptors in their model cell line, SW480. Reverse transcription was performed using the Reverse Transcription System and 1µg of total RNA isolated using the SV Total RNA Isolation System. The resulting cDNA (5µg) was amplified for 35 cycles (β-actin as a control) or 40 cycles (TrkA and p75NTR). RT-PCR results were confirmed by Western blot analysis.
(0003884) |
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Products: GoTaq® Green Master Mix | Reverse Transcription System | SV Total RNA Isolation System |
| 6. |
Wang, L., Zheng, G.G., Ma, C.H., Lin, Y.M., Zhang, H.Y., Ma, Y.Y., Chong, J.H. and Wu, K.F.
(2008)
A special linker between macrophage and hematopoietic malignant cells: membrane form of macrophage colony-stimulating factor.
Cancer Res.
68
,
5639–5647
.
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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.
(0003989) |
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Products: pTARGET™ Mammalian Expression Vector System |
| 7. |
Haraguchi, M., Okubo, T., Miyashita, Y., Miyamoto, Y., Hayashi, M., Crotti, T.N., McHugh, K.P. and Ozawa, M.
(2008)
Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins.
J. Biol. Chem.
283
,
23514–23
.
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Notes:
Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System.
(0003882) |
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Products: Dual-Luciferase® Reporter Assay System | GoTaq® DNA Polymerase | pGL3-Basic Vector |
| 8. |
Hou, Q., Wu, Y.H., Grabsch, H., Zhu, Y., Leong, S.H., Ganesan, K., Cross, D., Tan, L.K., Tao, J., Gopalakrishnan, V., Tang, B.L., Kon, O.L. and Tan, P.
(2008)
Integrative genomics identifies RAB23 as an invasion mediator gene in diffuse-type gastric cancer.
Cancer Res.
68
,
4623–4630
.
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Notes:
In this article, the researchers explored the role of RAB23 in gastric cancers. Twenty-four hours before transfection, AGS cells (gastric cancer cell line) that expressed RAB23 were seeded into a 24-well plate at a density of 1.8 × 105 cells/ml. To overexpress RAB23, a full-length RAB23 cDNA was cloned into the pCI-neo Mammalian Expression Vector and transfected into AGS cells. The effect of RAB23 overexpression on the cells was determined using a Matrigel invasion assay while protein expression levels were visualized with Western blotting.
(0003986) |
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Products: pCI-neo Mammalian Expression Vector |
| 9. |
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 |
| 10. |
Rambow-Larsen, A.A., Rajashekara, G., Petersen, E. and Splitter G.
(2008)
Putative quorum-sensing regulator BlxR of Brucella melitensis regulates virulence factors including the type IV secretion system and flagella.
J. Bacteriol.
190
,
3274–3282
.
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Notes:
To examine the role of the LuxR-type regulatory protein (BlxR) in Brucella melitensis, a blxR mutant was created. The bacteria were cultured and total RNA extracted. Using 7 µg of total RNA, cDNA was synthesized, labeled with Cy®Dye-3-dCTP and purified using the ChipShot™ Direct Labeling and Clean-Up System. The labeled cDNA was then used in microarray hybridization and analysis of expression.
(0003981) |
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Products: ChipShot™ Direct Labeling and Clean-Up System | ChipShot™ Direct Labeling System |
| 11. |
Rolfs, A., Montor, W.R., Yoon, S.S., Hu, Y., Bhullar, B., Kelley, F., McCarron, S., Jepson, D.A., Shen, B., Taycher, E., Mohr, S.E., Zuo, D., Williamson, J,. Mekalanos, J. and Labaer, J.
(2008)
Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae.
Proc. Natl. Acad. Sci. USA
105
,
4364-4369
.
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Notes:
These authors prepared a complete collection of Vibrio cholerae ORF clones using an automated amplification and cloning procedure. They then tested this set of clones for protein expression and capture using a nucleic acid programmable protein array method. To do this, they used the TNT® T7 Coupled Reticulocyte Lysate System to perform in situ transcription/translation of cDNA clones containing a GST fusion tag. Proteins were captured using an anti-GST antibody and subjected to further analysis.
(0003879) |
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Products: Bright-Glo™ Luciferase Assay System | TNT® T7 Coupled Reticulocyte Lysate System |
| 12. |
O'Reilly, K.C., Trent, S., Bailey, S.J. and Lane, M.A.
(2007)
13-cis-Retinoic acid alters intracellular serotonin, increases 5-HT1A receptor, and serotonin reuptake transporter levels in vitro.
Exp. Biol. Med.
232
,
1195–1203
.
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Notes:
The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers.
(0003790) |
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Products: Reverse Transcription System |
| 13. |
Tan, K.P., Yang, M. and Ito, S.
(2007)
Activation of Nrf2 by toxic bile acids provokes adaptive defense responses to enhance cell survival at the emergence of oxidative stress.
Mol. Pharmacol.
72
,
1380–1390
.
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Notes:
The authors explored the role that nuclear factor (erythroid 2-like) factor 2 (Nrf2) may have in mitigating the cytotoxic effects of bile acids on cells. A reporter vector was constructed using the core sequence of antioxidant reporter element (ARE) sythnesized by annealing two complementary oligonucleotides with Kpn1 and BglII at the 5’ and 3’ ends, respectively, and ligated into the same restriction sites of the pGL3-Promoter Vector (designated pGL3_ARE). To ensure specificity for the experiments, three point mutations were introduced into the ARE sequence (designated pGL3_mARE). To create HepG2 cells that stably expressed human Na(+)-taurocholate co-transporting polypeptide (NTCP), the cDNA clone of NTCP was subcloned into pTargeT™ Mammalian Expression Vector via the NotI site and selected using 500μg/ml G-418. The stable clones or standard HepG2 cells were transiently transfected with 0.1µg of pGL3_ARE or pGL3_mARE, 0.02µg of the control reporter pRL-TK Vector, with or without Nrf2 or dominant negative Nrf2 expression constructs. After overnight transfection, the cells were treated with bile acids for 16–18 hours and luciferase activities determined using the Dual-Luciferase® Reporter Assay System. Each experiment was done in triplicate and repeated at least two times.
(0003691) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Promoter Vector | pRL-TK Vector | pTARGET™ Mammalian Expression Vector System |
| 14. |
Clauser, S., Peyrard, S., Gaussem, P., Crespin, M., Emmerich, J., Aiach, M. and Borgel, D.
(2007)
Development of a novel immunoassay for the assessment of plasma Gas6 concentrations and their variation with hormonal status.
Clin. Chem.
53
,
1808–1813
.
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Notes:
To examine the effects of hormones on Gas6, a plasma vitamin K-dependent protein that may play a role in cardiovascular disease, the authors developed an ELISA test for Gas6, which they tested on blood from male and female volunteers. A recombinant Gas6 control was developed by reverse transcribing the full-length human Gas6 mRNA from human umbilical vein endothelial cells, amplifying the cDNA using nested PCR and after restriction digestion, ligating the insert into the EcoRI and XbaI sites of the pCI-neo Mammalian Expression Vector. The full-length construct was confirmed by sequencing and then tested in the Gas6 ELISA.
(0003688) |
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Products: pCI-neo Mammalian Expression Vector |
| 15. |
Dong, M., How, T., Kirkbride, K.C., Gordon, K.J., Lee, J.D., Hempel, N., Kelly, P., Moeller, B.J., Marks, J.R., and Blobe, G.C.
(2007)
The type III TGF-b receptor suppresses breast cancer progression.
J. Clin. Invest.
117
,
206-17
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Notes:
These authors showed that loss of the type II TGF-β receptor TGFβIII through allelic imbalance occurs during breast cancer development and increases metastatic potential. When TGFβIII expression was restored in human breast cancer cells, invasiveness, angiogenesis and metastasis were inhibited in an in vivo model system. The authors first analyzed TGFβIII mRNA levels using a cDNA array of 50 different breast cancer samples and controls. They also investigated TGFβIII protein expression levels by immunohistochemical analysis of a breast cancer tissue array containing over 250 breast cancers specimens at different stages of disease progression. Results from both analyses showed that TGFβIII expression decreased as disease progressed. The effect pf TGFβIII expression on tumor growth was investigated in a mouse model system. Murine 4T1 mammary cancer cells genetically engineered to express firefly luciferase were stably transfected with an expression vector containing TGFβIII , or a control vector. Tumor progression was then monitored in vivo by bioluminescent imaging. Cells expressing TGFβIII had delayed onset of metastasis and a reduction on the size and number of metastases compared with non-TGFβIII-expressing cells.
(0003617) |
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Products: Luciferin-EF™ |
| 16. |
Ghosh, S., Lu, Y., Katz, A., Hu, Y., and Li, R.
(2007)
Tumor suppressor BRCA1 inhibits a breast cancer-associated promoter of the aromatase gene (CYP19) in human adipose stromal cells.
Am. J. Physiol. Endocrinol. Metab.
292
,
E246-E252
.
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Notes:
Obesity-associated elevated estrogen increases the risk for breast cancer in postmenopausal women. The rate limiting step in the synthesis of estrogen from androgen is catalyzed by the aromatase enzyme. Normally this enzyme is expressed under a weak promoter in adipose tissue; however in breast cancer a second, strong ovary-specific promoter (PII) drives expression of aromatase. This study investigated the relationship of BRCA1 and aromatase expression. RNA isolated from BRCA-1 siRNA-treated adipose stromal cells was reverse transcribed using the ImProm-II™ Reverse Transcription System. The authors show that siRNA knockdown of BRCA1 resulted in activation of the PII promoter, suggesting that BRCA1 can modulate estrogen biosynthesis in adipose tissue.
(0003606) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 17. |
Witkowski, J.M., Soroczynska-Cybula, M., Bryl, E., Smolenska, Z., and Jozwik, A.
(2007)
Klotho—a Common Link in Physiological and Rheumatoid Arthritis-Related Aging of Human CD4 Lymphocytes
J. Immunology
178
,
771–777
.
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Notes:
Klotho knockout mice exhibit a phenotype of precocious aging, organ failure, osteoporosis. Humans with specific Klotho alleles are at increased risk for osteoporosis, atherosclerosis and decreased lifespan. The authors of this study looked at the expression of Klotho in CD4+ lymphocytes in patients suffering from rheumatoid arthritis and age-matched healthy individuals. cDNA was prepared from total RNA isolated from purified CD4+ lymphocytes using the ImProm-II™ Reverse Transcription System. Klotho expression, protein level and activity was decreased in the lymphocytes from the RA patients.
(0003654) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 18. |
Liu, Y., Kern, J.T., Walker, J.R., Johnson, J.A., Schultz, P.G., and Luesch, H.
(2007)
A genomic screen for activators of the antioxidant response element.
Proc. Natl. Acad. Sci. U S A
104
,
5205-5210
.
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Notes:
These authors screened a library of 15,000 expression cDNAs in neuroblastoma IMR-32 cells searching for genes that activate the antioxidant response element, ARE. ARE is a cis-acting enhancer element found in the 5´ flanking region of many genes that are involved in protection from oxidative stress. The library was screened using a luciferase reporter construct under the control of an ARE-containing promoter. Luminescence, indicating the presence of cDNA-activating ARE, was measured using the Bright-Glo™ Luciferase Assay System. cDNA clones showing reproducible activation were selected for further analysis. The authors tested the effect of over expression of these ARE activators on the ability to resist oxidative stress. IMR-32 cells expressing the various cDNAs were exposed to hydrogen peroxide or rotenone, and the effect on cell viability was measured using the CellTiter-Glo® Assay. Cells overexpressing the ARE-activators were more resistant to oxidative stress than controls.
(0003629) |
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Products: Bright-Glo™ Luciferase Assay System | CellTiter-Glo® Luminescent Cell Viability Assay |
| 19. |
Blommel, P.G., Martin, P.A., Wrobel, R.L., Steffen, E. and Fox, B.G.
(2006)
High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system.
Protein Expr. Purif.
47
,
562–570
.
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Notes:
In this study, the Flexi® Vector Systems was compared with the Gateway® Cloning System to determine its utility in high-throughput expression cloning by subcloning 96 human target genes. A direct comparison between pVP16, the Gateway® vector and the equivalent Flexi® Vector, pVP33A or K, was achieved by modifying pVP16 with the barnase gene and PmeI/SgfI restriction sites, duplicating the design available in the commercial Flexi® Vectors. Capture of genes by PCR amplification of the cDNAs was similar for both systems, but the timeline for the Flexi® Vector system was shorter at 6–8 days compared to 12 days for the Gateway® system. They also found the Flexi® Vector System was lower cost and more accurate due to the shorter primers required for the Flexi® Vector cloning. The authors found nearly twofold fewer missense errors due to the shorter amplification primers. Ninty-six cDNAs were amplified simultaneously in their protocol and PCR products were cleaned up using either the MagneSil® PCR Clean-Up System or Wizard® SV 96 PCR Clean-Up, ligated into the Flexi® Vector, and transformed into Select96™ Competent Cells. The study also compared transfer of cDNA inserts between different Flexi® Vectors and transfer of cDNA inserts between different Gateway® vectors and found similar performance in the two systems. For the Flexi® Vector test set, the authors sequenced the clones, validating the high fidelity transfer of cDNA inserts between Flexi® Vectors.
(0003533) |
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Products: Flexi® System, Entry/Transfer | Flexi® System, Transfer | Wizard® SV 96 PCR Clean-Up System |
| 20. |
Desfarages, S., San Filippo, J., Fournier, M. Calmels, C., Caumont-Sarcos, A., Litvak, S., Sung, P., Parissi, V.
(2006)
Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51
Nucleic Acids Res.
34
,
6215-6224
.
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Notes:
In the process of demonstrating the role of IN in HIV-1 integration in yeast, the authors purified all DNA vectors and PCR products with the Wizard® Plus SV Miniprep System and Wizard® SV Gel System. PCR products were generated using Taq DNA Polymerase. The pGEM®-T Vector was used to clone amplification products. Sequencing was performed using BamHI, religated with T4 DNA Ligase.
(0003704) |
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Products: BamHI | GoTaq® DNA Polymerase | pGEM®-T Vector System I | T4 DNA Ligase | Wizard® Plus SV Minipreps DNA Purification System | Wizard® SV Gel and PCR Clean-Up System |
| 21. |
Pandhare, J., Cooper, S.K. and Phang, J.M.
(2006)
Proline oxidase, a proapoptotic gene, is induced by troglitazone: evidence for both peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms.
J. Biol. Chem.
281
,
2044–2052
.
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Notes:
A proline oxidase (POX) antisense vector was generated by amplifying part of the POX cDNA and ligating the product into the pCI Mammalian Expression Vector in the antisense orientation. This construct was tested and validated for blocking POX mRNA expression using RT-PCR. Both PPARγ and p53 cDNAs were also cloned into the pCI Vector. The human POX promoter sequence was amplified and cloned into the NheI and HindIII sites of the pGL3-Basic Vector to create the POX-Luc reporter construct. Using several colon cancer cell lines (HT29, LoVo, HCT116, HCT15, RKO, KM12, HCC2998 and SW620), the POX-Luc construct was co-transfected with pRL-null (to normalize transfection efficiency) plus PPARγ, p53 contructs or empty vector. A PPARγ ligand was added 10 hours post-transfection and cells harvested 24–36 hours after transfection. POX promoter luciferase activity was measured using the Dual-Luciferase® Reporter Assay System and a TD-20/20 luminometer.
(0003514) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pCI-neo Mammalian Expression Vector | pGL3-Basic Vector | pRL-null Vector |
| 22. |
de Wolf, C.J., Cupers, R.M., Bertina, R.M. and Vos, H.L.
(2006)
The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter.
J. Biol. Chem.
281
,
17635–17643
.
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Notes:
The Protein S promoter (PROS1) fragment –5948/–1 was cloned directly 5’ to the firefly luciferase reporter gene in the pGL3-Basic Vector using the KpnI and XhoI enzyme sites. This construct, PS5948-luc, was linearized with KpnI and NdeI and subsequently subjected to progressive deletion using the Erase-a-Base® System. The size of the resulting 5’-deletion was determined by sequence analysis, and the deletion constructs were used for transient transfection assays. HepG2, HuH7, HeLa and HUVEC cells were transfected at 60–80% confluency in 12-well plates using 3µl of Tfx™-20 per microgram DNA. In each transfection, an equimolar concentration of construct was used and supplemented with an additional plasmid to keep the amount of transfected DNA constant. pRL-SV40 Vector was co-transfected as a transfection control using a 1:500 ratio to the total transfected amount of DNA in HepG2, HuH7 and HeLa cell lines, and a 1:100 ratio in transfections with HUVEC and 1 × 106 Meg01 suspension cells. Transcription factor expression vector (250ng) was co-transfected, and expression vector without the transcription factor cDNA was used as a negative control. Cell extracts were harvested at either 24 (HepG2 and HuH7) or 48 hours (Meg01, HUVEC, and HeLa) post-transfection using 250µl of Passive Lysis Buffer per well. Luciferase activity was determined using 20–100µl of lysate with the Dual-Luciferase® Reporter Assay System.
(0003510) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Erase-a-Base® System (minus vectors & bacterial strain) | pGL3-Basic Vector | Tfx™-20 Reagent |
| 23. |
Wijeratne, A.J., Chen, C., Zhang, W., Timofejeva, L. and Ma, H.
(2006)
The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination.
Mol. Cell Biol.
17
,
1331-1343
.
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Notes:
The authors identify PARTING DANCERS as a gene involved in male meiosis in Arabidopsis using a microarray generated from meiotic-stage anthers. To confirm the sequence obtained by microarray analysis, RT-PCR was performed and the resulting cDNA was cloned into the pGEM®-T Vector and sequenced. To generate probes for in situ hybridization, fragments of ptd were amplified, cloned into the pGEM®-T Vector, then labeled with digoxygenin through in vitro transcription.
(0003468) |
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Products: pGEM®-T Vector System I | pGEM®-T Vector System II |
| 24. |
Lang, C., Schulze, J., Mendel, R-R. and Hänsch, R.
(2006)
HaloTag™: A new versatile reporter gene system in plant cells.
J. Exp. Bot.
July
,
epub ahead of print
.
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Notes:
This paper highlights the first use of the HaloTag™ Interchangeable Protein Labeling Technology in plant cells. The cDNA for the HaloTag™ protein was amplified by PCR from the pHT2 Vector and cloned into the pGEM®-T Easy® Vector, from which it was excised and transferred to a second vector where its expression was under the control of the cauliflower mosaic virus (CaMV)-35S promoter. The construct was transformed into tobacco protoplasts and bioballistically transformed into tobacco leaf cells. Localization was followed using the HaloTag™ TMR and diAcFAM Ligands.
(0003503) |
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Products: HaloTag® pHT2 Vector | HaloTag® TMR Ligand | pGEM®-T Easy Vector System I |
| 25. |
Temple, G., Lamesch, P., Milstein, S., Hill, D.E., Wagner, L., Moore, T. and Vidal, M.
(2006)
From genome to proteome: developing expression clone resources for the human genome.
Hum. Mol. Genet.
15
,
R31-R43
.
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Notes:
The Kazusa cDNA Project is constructing a library of more than 1,000 "full ORF" (F-ORF) clones in the Flexi® Vector System to characterize the function of proteins that are larger than 50kD.
(0003393) |
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Products: pF1A T7 Flexi® Vector | pF1K T7 Flexi® Vector | pF3A WG (BYDV) Flexi® Vector | pF3K WG (BYDV) Flexi® Vector | pF4A CMV Flexi® Vector | pF4K CMV Flexi® Vector | pFC7A (HQ) Flexi® Vector | pFC7K (HQ) Flexi® Vector | pFC8A (HaloTag®) CMV Flexi® Vector | pFC8K (HaloTag®) CMV Flexi® Vector | pFN2A (GST) Flexi® Vector | pFN2K (GST) Flexi® Vector | pFN6A (HQ) Flexi® Vector | pFN6K (HQ) Flexi® Vector |