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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. |
Izukawa, T., Nakajima, M., Fujiwara, R., Yamanaka, H., Fukami, T., Takamiya, M., Aoki, Y., Ikushiro, S., Sakaki, T. and Yokoi, T.
(2009)
Quantitative analysis of UDP-glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers.
Drug Metab. Dispos.
37
,
1759–1768
.
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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.
(0004034) |
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Products: pTARGET™ Mammalian Expression Vector System | pTARGET™ Vector |
| 3. |
Terui, Y., Mishima, Y., Sugimura, N., Kojima, K., Sakurai, T., Mishima, Y., Kuniyoshi, R., Taniyama, A., Yokoyama, M., Sakajiri, S., Takeuchi, K., Watanabe, C., Takahashi, S., Ito, Y. and Hatake, K.
(2009)
Identification of CD20 C-terminal deletion mutations associated with loss of CD20 expression in non-Hodgkin's lymphoma.
Clin. Cancer Res.
15
,
2523–2530
.
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Notes:
The researchers examined if a CD20 mutation would affect resistance to rituximab, an adjuct cancer therapy drug used for CD20-positive B-cell lymphoma. CD20 PCR products amplified from genomic DNA were cloned into the pTARGET™ Mammalian Expression Vector. These CD20 mutant constructs were stably introduced into K562 chronic myelogenous leukemia cells by electroporation and selected using G-418. One microgram of CD20 mutant construct DNA was transcribed and translated using an in vitro translation kit from Promega.
(0004032) |
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Products: pTARGET™ Mammalian Expression Vector System | pTARGET™ Vector |
| 4. |
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 |
| 5. |
Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.
(2008)
Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products.
DNA Research
15
,
137-149
.
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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.
(0003800) |
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Products: Anti-HaloTag® pAb | Dual-Glo® Luciferase Assay System | Flexi® System, Entry/Transfer | Flexi® System, Transfer | GloMax® 96 Microplate Luminometer w/Dual Injectors | Monster Green® Fluorescent Protein phMGFP Vector | pF1K T7 Flexi® Vector | pGL4.10[luc2] Vector | TNT® SP6 High-Yield Wheat Germ Protein Expression System | T7 RiboMAX™ Express Large Scale RNA Production System | Wheat Germ Extract Plus | Wizard® SV 96 PCR Clean-Up System |
| 6. |
Leseberg, C.H., Eissler, C.L., Wang, X., Johns, M.A., Duvall, M.R. and Mao, L.
(2008)
Interaction study of MADS-domain proteins in tomato.
J. Exp. Bot.
59
,
2253–65
.
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Notes:
The authors characterized the network of protein-protein interactions for 22 MADS-domain proteins in tomato using yeast two-hybrid and three-hybrid assays. To construct bait and prey proteins, total RNA from various tissues was reverse transcribed using the Reverse Transcription System, then amplified using PCR primers containing restriction enzyme sites for cloning into the bait and prey vectors.
(0003886) |
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Products: Reverse Transcription System |
| 7. |
Dall'Osso, C., Guella, I., Duga, S., Locatelli, N., Paraboschi, E.M., Spreafico, M., Afrasiabi, A., Pechlaner, C., Peyvandi, F., Tenchini, M.L. and Asselta, R.
(2008)
Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.
Haematologica
93
,
1505–1513
.
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Notes:
To examine the causes of Factor V (FV) deficiency, the authors examined transcript splicing and its mutated variations. Three regions of human FV (F5) were amplified from a healthy individual and the PCR products cloned into the pTargeT™ Mammalian Expression Vector. Three identified mutations from people with FV deficiency were introduced by site-directed mutatgenesis. All constructs were sequenced before transfection into HeLa cells. After 48 hours, the total RNA was purified and the splicing pattern of the wild type and mutant constructs were analyzed by RT-PCR. The mutant constructs were also transfected into HepG2 cells and tested for nonsense-mediated mRNA decay (NMD) with or without NMD inhibitors (puromycin, cycloheximide, and wortmannin) using RT-PCR.
(0003992) |
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Products: pTARGET™ Mammalian Expression Vector System |
| 8. |
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 |
| 9. |
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 |
| 10. |
Kurth, E.G., Doughty, D.M., Bottomley, P.J., Arp, D.J. and Sayavedra-Soto, L.A.
(2008)
Involvement of BmoR and BmoG in n-alkane metabolism in 'Pseudomonas butanovora'.
Microbiology
154
,
139–47
.
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Notes:
The authors characterized five open-reading frames flanking the alcohol-inducible alkane monooxygenase (BMO) structural gene of Pseudomonas butanovora. Strains with mutated bmoR, which encodes a putative transcriptional regulator, or bmoG, which encodes a putative chaperonin, were created by gene inactivation. The bmoR gene was amplified and cloned into the pGEM®-T Vector for disruption with a kanamycin cassette. The two termini of the bmoG gene were amplified separately, ligated to the kanamycin cassette and cloned into the pGEM®-T Easy Vector. Plasmids encoding the disrupted genes were transformed into Pseudomonas butanovora by electroporation.
(0003893) |
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Products: pGEM®-T Easy Vector System I | pGEM®-T Vector System I |
| 11. |
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 |
| 12. |
Takahashi, T., Murakami, K., Nagakura, M., Kishita, H., Watanabe, S., Honke, K., Ogura, K., Tai, T., Kawasaki, K., Miyamoto, D., Hidari, K.I., Guo, C.T., Suzuki, Y. and Suzuki, T.
(2008)
Sulfatide is required for efficient replication of influenza A virus.
J. Virol.
12
,
5940–50
.
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Notes:
Sulfatide is present in mammalian organs where influenza A replicates. Ceramide galactosyltransferase (CGT) and cerebroside (galactosylceramide) sulfotransferase (CST), which synthesize sulfatide, were cloned by PCR into the pTargeT™ Mammalian Expression Vector and the pGEM®-T Easy Vector, (CST with or without a three base insertion), respectively. The two genes were removed by restriction digestion and cloned into pIRES-neo to forma bicistronic construct. Arylsulfatase A (ASA), which degrades sulfatide was also amplified and cloned into the pGEM®-T Easy Vector, before being sublconed into a neomycin-resistant expression vector. The expression vectors were transfected into COS-7 cells and selected for stable expression using G418.
(0003990) |
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Products: Antibiotic G-418 Sulfate | Antibiotic G-418 Sulfate Solution | pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | pTARGET™ Mammalian Expression Vector System |
| 13. |
Purbey, P.K., Singh, S., Kumar, P.P., Mehta, S., Ganesh, K.N., Mitra, D. and Galande, S.
(2008)
PDZ domain-mediated dimerization and homeodomain-directed specificity are required for high-affinity DNA binding by SATB1.
Nucleic Acids Res.
36
,
2107–2722
.
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Notes:
To learn about the ideal target binding sequence for SATB1, the T-lineage-enriched chromatin organizer and transcription factor, random oligonucleotides underwent SELEX and five rounds of selection by EMSA. The enriched library of oligos was cloned into the pGEM®-T Easy Vector, transformed and sequenced. Several variants of SATB1-binding consensus sequences were annealed, ligated into the pGL3-Promoter Vector and cotransfected into HEK 293 cells with a plasmid that either contained SATB1 or was empty. After 48 hours, the cells were harvested and luciferase activity measured. The CheckMate™ Mammalian Two-Hybrid System was used to assess how the N-terminal PDZ domain of SATB1 interacted with the Cut and homeodomain in the C-terminus.
(0003982) |
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Products: CheckMate™ Mammalian Two-Hybrid System | pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | pGL3-Promoter Vector |
| 14. |
Abdel-Latief, M., Garbe, L.A., Koch, M., and Ruther, J.
(2008)
An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site.
Proc. Natl. Acad. Sci. USA
105
,
8914-8919
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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.
(0003903) |
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Products: pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | PureYield™ Plasmid Midiprep Start-Up Kit, 220V Electrical (100 preps, manifold, 4 elution devices & free vacuum pump) | PureYield™ Plasmid Midiprep Start-Up Kit, 230V Electrical (100 preps, manifold, 4 elution devices & free vacuum pump) | PureYield™ Plasmid Midiprep System | Wizard® SV Gel and PCR Clean-Up Start-Up Kit, 110V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Gel and PCR Clean-Up Start-Up Kit, 220V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Gel and PCR Clean-Up Start-Up Kit, 230V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Gel and PCR Clean-Up System |
| 15. |
Cheung, Q.C., Turner, P.V., Song, C., Wu, D., Cai, H.Y., MacInnes, J.I. and Li, J.
(2008)
Enhanced resistance to bacterial infection in protegrin-1 transgenic mice.
Antimicrob. Agents Chemother.
52
,
1812–9
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Notes:
One potential source of antibiotic-resistant bacteria is food-producing animals. The authors examined the ability of protegrin-1 (PG-1), an antimicrobial peptide, to protect wildtype and transgenic mice expressing PG-1 against bacterial infection. As part of the cloning strategy to produce the PG-1 expression construct, the authors amplified and cloned full-length PG-1 into the pGEM®-T Easy Vector. To test the bactericidal activity of PG-1 expressed in transgenic mice, radial diffusion assays were performed, in which test samples were added to a well containing E. coli and the clear antibacterial zone was measured. Two of the test samples were neutrophil secretions from the PG-1 transgenic mice and purified polyhistidine-tagged PG-1 protein, purified using the MagneHis™ Protein Purification System.
(0003896) |
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Products: MagneHis™ Protein Purification System | pGEM®-T Easy Vector System I |
| 16. |
Bröker, D., Dietz, D., Arenskötter, M. and Steinbüchel, A.
(2008)
The genomes of the non-clearing-zone-forming and natural-rubber-degrading species Gordonia polyisoprenivorans and Gordonia westfalica harbor genes expressing Lcp activity in Streptomyces strains.
Appl. Environ. Microbiol.
74
,
2288–97
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Notes:
Natural rubber-degrading bacteria fall into two categories: those forming clearing zones on latex overlay plates and those that do not. To investigate this degradation process, the authors amplified latex-clearing protein (lcp) homologs from non-clearing-zone-forming bacteria using degenerate PCR primers based on lcp sequences from clearing-zone forming species. The 3´ region of the lcp gene in G. westfalica was amplified by nested PCR using biotinylated primers, and the amplified products were cloned in the pGEM®-T Easy Vector and sequenced using universal M13 forward and reverse primers.
(0003907) |
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Products: pGEM®-T Easy Vector System I |
| 17. |
Jin, Z., Lahat, G., Korchin, B., Nguyen, T., Zhu, Q.S., Wang, X., Lazar, A.J., Trent, J., Pollock, R.E. and Lev D.
(2008)
Midkine enhances soft-tissue sarcoma growth: a possible novel therapeutic target.
Clin. Cancer Res.
14
,
5033–42
.
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Notes:
Increased expression of midkine (MK), a growth factor normally involved in neural development, is associated with several human malignancies. The authors used quantitative RT-PCR to examine mRNA levels for MK and MK receptors in various soft-tissue sarcoma (STS) cell lines. Reverse transcription was performed using 1µg of total RNA, and 2µl of cDNA was used in qPCR using the PCR Master Mix, primers specific to MK and either glyceraldehyde-3-phosphate or β-actin primers for normalization, and EvaGreen® dye. To examine the protumorigenic effects of MK, the authors incubated HT1080 and SW684 STS cell lines, which express low levels of MK, with human recombinant MK and measured cell proliferation using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. In addition, the authors examined cell proliferation in MK-stably transfected HT1080 cells. The plasmid used for stable transfections was created by reverse transcribing the MK-coding region using the ImProm-II™ Reverse Transcription System, then cloning the resulting cDNA into an expression vector.
(0003895) |
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Products: CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay |
| 18. |
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
.
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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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| 19. |
Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.
(2007)
Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells.
Mol. Cell. Endocrinol.
264
,
50-60
.
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Notes:
This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms.
(0003618) |
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Products: Caspase-Glo® 3/7 Assay | CellTiter-Blue® Cell Viability Assay | ImProm-II™ Reverse Transcriptase | M-MLV Reverse Transcriptase Buffer Pack | M-MLV Reverse Transcriptase, RNase H Minus | pTARGET™ Mammalian Expression Vector System |
| 20. |
Renaud, S., Loukinov, D., Abdullaev, Z., Guilleret, I., Bosman, F.T., Lobanenkov, V. and Benhattar, J.
(2007)
Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene
Nucleic Acids Research
35
,
1245-1256
.
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Notes:
Telomeres shorten by 50–100 bases with each cell division, making the telomere a "mitotic counter" that can limit cellular lifespan. Telomerase is a two-component protein consisting of a reverse transcriptase (hTERT) bound to its own RNA template that can act to maintain telomere length in dividing cells. Telomerase is highly active in dividing cells such as germ cells, stem cells and many cancers. This paper investigated the role of methylation of the hTERT promoter and the transcription factor CTCF in regulation of telomerase activity. LacZ reporter plasmids driven by the hTERT minimal promoter were transiently transfected into HeLa cells, and reporter assays were performed on lysate generated using Passive Lysis Buffer. The hTERT minimal promoter did not show activity if all of the CpG sites were methylated. The promoter and first exon of hTERT were amplified using PCR Master Mix from sodium bisulfite-treated genomic DNA isolated from telomerase-positive cell lines and tissues. The resulting fragments were cloned using the pGEM®-T Vector System II. For the methylation cassette assay, methylated and unmethylated fragments were cloned into a methylated or unmethylated vector using the LigaFast™ Rapid DNA Ligation System. The authors conclude that methylation plays a dual role in regulating hTERT expression. CTCF will bind to the first exon of hTERT when the hTERT CpG island is not methylated, resulting in downregulation of hTERT expression. Although CTCF cannot bind the hTERT promoter when the DNA is completely methylated, the methylation itself completely represses transcription. In situations where there is partial methylation of the promoter, such as in tumor cells, CTCF cannot bind to the promoter, but the partial methylation is not enough to repress transcription, and hTERT is expressed.
(0003641) |
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Products: LigaFast™ Rapid DNA Ligation System | MspI | Passive Lysis 5X Buffer | PCR Master Mix | pGEM®-T Vector System II |
| 21. |
Rendón, M.A., Saldaña, Z., Erdem, A.L., Monteiro-Neto, V., Vázquez, A., Kaper, J.B., Puente, J.L. and Girón, J.A.
(2007)
Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization.
Proc. Natl. Acad. Sci. USA.
104
,
10637–10642
.
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Notes:
The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector cotaining the ecpA gene, restored the strain's ability to adhere to epithelial cells.
(0003719) |
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Products: GoTaq® Green Master Mix | pGEM®-T Vector System I |
| 22. |
Cheng, X., Liu, J., Asuncion-Chin, M., Blaskova, E., Bannister, J.P., Dopico, A.M. and Jaggar, J.H.
(2007)
A novel CaV1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits.
J. Biol. Chem.
282
,
29211–21
.
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Notes:
The authors identified a novel subunit of the voltage-dependent L-type Ca2+ channel (CaV1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM®-T Easy Vector and sequenced using the T7 Promoter Primer.
(0003801) |
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Products: pGEM®-T Easy Vector System I | T7 Promoter Primer |
| 23. |
Valiante, S., Prisco, M., Capaldo, A., Zambrano, I., De Falco, M., Andreuccetti, P., Laforgia, V., and Varano, L.
(2007)
Molecular characterization and gene expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the lizard brain.
Brain Res.
1127
,
66–75
.
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Notes:
The authors cloned pituitary adenylate cyclase-activating polypeptide (PACAP) from lizard (Podarcis sicula) brain. They then isolated total RNA from lizard brain using the SV Total RNA Isolation System and used 4µg of total RNA in a reverse transcription with ImProm-II™ Reverse Transcriptase and oligo(dT)15 primers at 37°C for 1.5 hours. The PACAP cDNA was amplified by PCR, and the resulting PCR products were cleaned up using the Wizard® SV Gel and PCR Clean-Up System prior to sequencing.
(0003666) |
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Products: ImProm-II™ Reverse Transcriptase | SV Total RNA Isolation System | Wizard® SV Gel and PCR Clean-Up System |
| 24. |
Brown, K.A., Boerboom, D., Bouchard, N., Doré, M., Lussier, J.G. and Sirois, J.
(2007)
Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo.
J. Mol. Endocrinol.
36
,
449–461
.
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Notes:
The authors cloned the novel equine aldo-keto reductase AKR1C23 and characterized its expression patterns in the preovulatory follicle. The AKR1C23 cDNA was amplified from equine ovarian RNA using the Access RT-PCR System and primers designed by sequence alignments of known AKR sequences, then cloned into the pGEM®-T Easy Vector. Levels of AKR1C23 and ribosomal protein L17a mRNAs in various equine tissues were quantified using the Access RT-PCR System and 21 cycles and 18 cycles, respectively, followed by agarose gel electrophoresis, transfer to nylon membranes, and hybridization to radiolabeled probes synthesized using the Prime-a-Gene® Labeling System.
(0003791) |
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Products: Access RT-PCR System | pGEM®-T Easy Vector System I | Prime-a-Gene® Labeling System |
| 25. |
Suzuki, A., Ogura, T. and Esumi, H.
(2006)
NDR2 acts as the upstream kinase of ARK5 during insulin-like growth factor-1 signaling.
J. Biol. Chem.
281
,
13915–13921
.
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Notes:
A deletion mutation of the serine/threonine protein kinase NDR2 was created by PCR using two mutagenesis primers and two plasmid-based primers. After amplification, the two products were run on a 1% agarose gel and extracted using the Wizard® SV Gel and PCR Clean-Up System. The purified fragments were mixed, annealed, re-amplified and then digested prior to cloning into an expression vector. The human colorectal cancer cell lines HCT-116, DLD-1, and SW480 used in the study were seeded into a 24-well plate at 5 × 104/well, and transfected using the TransFast™ Transfection Reagent. The transfection was assessed with a green fluorescent protein expression vector.
(0003438) |
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Products: TransFast™ Transfection Reagent | Wizard® SV Gel and PCR Clean-Up System |