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Clin. Can. Res. 15, 2523–2530. Identification of CD20 C-terminal deletion mutations associated with loss of CD20 expression in non-Hodgkin's lymphoma. 2009

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.

Notes: The researchers examined if a CD20 mutation would affect resistance to rituximab, an adjunct 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. (4032)

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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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Proc. Natl. Acad. Sci. USA 106, 2441–2446. Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct. 2009

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.

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. (4033)

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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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Haematologica 93, 1505–1513. Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern. 2008

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.

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. (3992)

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J. Virol. 12, 5940–50. Sulfatide is required for efficient replication of influenza A virus. 2008

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.

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 subcloned into a neomycin-resistant expression vector. The expression vectors were transfected into COS-7 cells and selected for stable expression using G418. (3990)

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Mol. Pharmacol. 72, 1380–1390. Activation of Nrf2 by toxic bile acids provokes adaptive defense responses to enhance cell survival at the emergence of oxidative stress. 2007

Tan, K.P., Yang, M. and Ito, S.

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. (3691)

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J. Biol. Chem. 282, 19619–19628. EphrinA1 activates a Src/focal adhesion kinase-mediated motility response leading to rho-dependent actino/myosin contractility. 2007

Parri, M., Buricchi, F., Giannoni, E., Grimaldi, G., Mello, T., Raugei, G., Ramponi, G. and Chiarugi, P.

Notes: This paper explored the process by which the Eph receptor EphA2 regulates repulsive response in prostatic carcinoma cells via the ephrinA1 ligand. Focal adhesion kinase (FAK) non-related kinase (FRNK; a target in the ephrinA1 ligand cascade) was subcloned into the pTargeT™ Mammalian Expression Vector. This mutant FAK was transfected into PC-3 cells and subjected to a GST-pulldown assay to examine RhoA activation. In addition, PC-3 cells transfected with several targets in the EphA2/ephrinA1 activation cascade including FRNK were subjected to an in vitro three-dimensional migration assay. (3690)

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Mol. Cell. Endocrinol. 264, 50-60. Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells. 2007

Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.

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. (3618)

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Am. J. Pathol. 168, 706–713. Expression of Pax2 in human renal tumor-derived endothelial cells sustains apoptosis resistance and angiogenesis. 2006

Fonsato, V., Buttiglieri, S., Deregibus, M.C., Puntorieri, V., Bussolati, B. and Camussi, G.

Notes: To further understand the transcription factor paired-box 2 (PAX2) gene, 2µg of total RNA isolated from Kaposi’s sarcoma cells were reverse transcribed, and 5µl of the RT reaction was amplified. The PCR product was then cloned into the pTARGET™ Mammalian Expression Vector and two clones identified: PAX2 in the sense orientation and a second in the antisense orientation. The clones were then transfected into two different cell lines: the antisense PAX2 into renal tumor-derived endothelial cells where PAX2 is expressed, and the sense into normal human microvascular endothelial cells where PAX2 is not normally expressed. The effects of PAX2 expression on the cell lines were then examined. (3496)

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Genetics 173, 2143–2149. Functional Implication of an ARG307GLY Substitution in Corticosteroid Binding Globulin, a Candidate Gene for a QTL Associated with Cortisol Variability and Obesity. 2006

Guyonnet-Duperat, V., Geverink, N., Plastow, G.S., Evans, G., Ousova, O., Croisetiere, C., Foury, A., Richard, E., Mormede, P. and Moisan, M.P.

Notes: In this study, the effects of amino acid substitutions in porcine corticosteroid-binding globulin gene (Cbg) were tested on CBG binding and affinity. Genomic DNA was isolated from whole blood of 92 female pigs studied. Cbg cDNA was obtained by reverse transcribing pig liver total RNA using M-MLV Reverse Transcriptase followed by PCR. The 1257pb cDNA PCR product was ligated into the pTARGET™ Mammalian Expression Vector. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce four different codon substitutions in the Cbg cDNA. Once created, the mutated and unmodified Cbg cDNA constructs were transfected into HEK-293T (human embryonic kidney) cells. After 48 hours, the supernatant was collected to analyze secreted CBG. (3498)

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Cancer Res. 66, 9090-9098. MicroRNA regulates the expression of human cytochrome P450 1B1. 2006

Tsuchiya, Y., Nakajima, M., Takagi, S., Taniya, T., and Yokoi, T.

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

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J. Biol. Chem. 281, 8254–8263. Molecular cloning and characterization of UDP-glucose dehydrogenase from the amphibian Xenopus laevis and its involvement in hyaluronan synthesis. 2006

Vigetti, D., Ori, M., Viola, M., Genasetti, A., Karousou, E., Rizzi, M., Pallotti, F., Nardi, I., Hascall, V.C., De Luca, G. and Passi, A.

Notes: To test the effect that Xenopus laevis UDP-glucose dehydrogenase (xUGDH) expression has in mammalian cells, the xUGDH ORF was amplified, purified, A-tailed and cloned into the pTARGET™ Mammalian Expression Vector. Two clones were selected: one in the sense orientation and one in the antisense orientation. The constructs were confirmed by DNA sequencing and expression in a TNT® T7 in vitro transcription/translation system. Five micrograms of the plasmids were transfected into AoSMCs and human aortic smooth muscle cells, and UGDH activities werw measured 48 hours post-transfection. (3497)

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Proc. Natl. Acad. Sci. USA 99, 13723-13728. A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity. 2002

Bufler, P., Azam, T., Gamboni-Robertson, F., Reznikov, L.L., Kumar, S., Dinarello, C.A., and Kim, S.-H.

Notes: The cDNA for the IL-1F7b protein was amplified from a human spleen cDNA library and directly subcloned in the pGEM®- T Easy Vector and sequenced. The clone was transferred to a bacterial expression vector with a purification tag. The expressed protein was used to make a polyclonal rabbit antibody and antibody purification column. The cDNA for the IL-1F7b was reamplified and directly subcloned into the pTARGET™ Mammalian Expression Vector. The cDNA was stably expressed in RAW264.7 mouse monocyte/macrophage cell line.  Lysates from the cells stably expressing IL-1F7b were used to test the antibodies by western blotting. Expressing cells were also tested by immunocytochemistry. (2598)

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Oncogene 20, 1041-1051. Isolation and characterization of the TERE1 gene, a gene down-regulated in transitional cell carcinoma of the bladder 2001

McGarvey, T.W., Nguyen, T., Tomaszewski, J.E., Monson, F.C. and Malkowicz, S.B.

Notes: The cDNA for the TERE1 gene was amplified by PCR and directly subcloned into the pTARGET™ Mammalian Expression Vector. The 338-amino acid (36.8kDa) TERE1 protein was stably expressed in three human bladder transitional cell carcinomas (J82, T24 and 1376 TCC) and the human embryonic kidney cell line, HEK 293. Stable transfectants were selected with the antibiotic G-418. Overexpression of the protein caused 80-90% inhibition of cellular proliferation in two of the transitional cell carcinoma cell lines and inhibition of aneuploidy in the third. Transfections were accomplished in 24-well plates with the Tfx™-20 Transfection Reagent. Excellent details of the 1 hour transfection protocol are presented. (2596)

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Oncogene 20, 260-269. Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, apoptotic body formation and phosphatidylserine externalization 2001

Droin, N., Rebe, C., Bichat, F., Hammann, A., Bertrand, R., and Solary, E.

Notes: The cDNA for the short isoform of the caspase-2 mRNA was amplified and cloned directly into the pTARGET™ Mammalian Expression Vector. The protein was stably expressed in U937 human leukemic cells by selection with the antibiotic G-418.  Stable clones were identified by PCR of the genomic DNA of selected colonies with primers to the T7 promoter of the pTARGET™ Vector and the downstream primer of the caspase-2 message. Overexpression of the protein interfered with etoposide-mediated apoptosis. (2597)

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J. Biol. Chem. 275, 6153-6159. ARA9 modifies agonist signaling through an increase in cytosolic aryl hydrocarbon receptor. 2000

LaPres, J.J., Glover, E., Dunham, E.E., Bunger, M.K., Bradfield, C.A.

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express a portion of the ARA9, a protein that interacts with the aryl hydrocarbon receptor. The expressed protein was used in a two-hybrid assay using a GAL4-fusion protein containing a portion of the aryl hydrocarbon receptor. Interaction of the two proteins is reported by activation of the luciferase in the pG5-luc Vector, which is a portion of the CheckMate™ Mammalian Two-Hybrid System. The two-hybrid studies were performed in COS-1 cells and were controlled with a cotransfection with a β-galactosidase vector. Cells were lysed with the Passive Lysis Buffer and luciferase activities determined with the Luciferase Assay System. (0842)

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J. Immunol. 164, 3961-3970. Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing Ligand (TRAIL) in human melanoma cells 2000

Zhang, X.D., Franco, A.V., Nguyen, T., Gray, C.P., Hersey, P.

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express the TRAIL-3 and the TRAIL-4 receptors. The proteins were expressed in primary human melanoma cells. (0098)

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Genes Dev. 14, 1353-1363. Dimerization and nuclear entry of mPER proteins in mammalian cells. 2000

Yagita, K., Yamaguchi, S., Tamanini, F., van Der Horst, G.T., Hoeijmakers, J.H., Yasui, A., Loros, J.J., Dunlap, J.C. and Okamura, H.

Notes: Three proteins were amplified with an HA tag and cloned into the pTargeT™ Mammalian Expression Vector for expression in transfected cells. The proteins were expressed in COS-7 cells, and were used to study protein association in cell extracts and in intact cells. (2153)

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J. Biol. Chem. 275, 11666-11671. Distinct roles for recombinant cytosolic 5'-nucleotidase-I and -II in AMP and IMP catabolism in COS-7 and H9c2 rat myoblast cell lines 2000

Sala-Newby, G.B., Freeman, N.V.E., Skladanowski, A.C., Newby, A.C.

Notes: The pTargeT™ Mammalian Expression Vector System was used to clone and express the human cytosolic 5'-nucleotidase II, a 65kDa protein. The protein was expressed and assayed in COS-7 monkey kidney cells and H9c2 rat myoblast cells. The pSV-beta Galactosidase Control Vector was used as a control for transfection efficiency. (0431)

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Nat. Cell Biol. 2, 757-761. Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation. 2000

Griffin, C.S., Simpson, P.J., Wilson, C.R., and Thacker, J.

Notes: The XRCC3 gene was amplified from a human testis cDNA library and directly subcloned into the pTARGET™ Mammalian Expression Vector. The insert was sequenced, then electroporated into the irs1SF hamster radiation-sensitive cell line. Stable transfectants were isolated by G-418 selection and used in chromosome segregation studies. (2599)

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J. Immunol. 164, 2248-2254. Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rapid identification of pathogenic TCR. 2000

Matsumoto, Y., Jee, Y. and Sugisaki, M.

Notes: The T-cell receptor of interest was amplified and subcloned in the pTARGET™ Mammalian Expression Vector. Large amounts of the vector were purified with the Wizard® Plus MegaPreps DNA Purification System and used as a DNA vaccine in Lewis rats. (0701)

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Hum. Gene Ther. 11, 247-261. The CXC chemokine, monokine-induced by interferon-gamma, inhibits non-small cell lung carcinoma tumor growth and metastasis 2000

Addison, C.L., Arenberg, D.A., Morris, S.B., Xue, Y.-Y., Burdick, M.D., Mulligan, M.S., Iannettoni, M.D. and Strieter, R.M.

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express MIG (monokine-induced by Interferon-gamma) and IP-10 (interferon-inducible protein 10) in A549 human adenocarcinoma cells. Stable transfectants were obtained through G418 selection. (2058)

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J. Biol. Chem. 274, 29720-29725. Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells. 1999

Halim, A.-B., LeGros, L., Geller, A., Kotb, M.

Notes: Subunits of the cDNA for methionine adenosyltransferase were amplified from a plasmid using Taq DNA Polymerase and directly cloned into the pTARGET™ Mammalian Expression T-Vector. The sequence of the cloned inserts were confirmed with the fmol® DNA Cycle Sequencing System. The constructs were transiently transfected into COS-1 cells with the TransFast™ Reagent at a 1:1 ratio. Excellent detail is provided for the transfection. (1094)

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J. Biol. Chem. 274, 7982-7986. KSR-1 binds to G-protein beta gamma subunits and inhibits beta gamma-induced mitogen-activated protein kinase activation. 1999

Bell, B., Xing, H., Yan, K., Gautam, N. and Muslin, A.J.

Notes: The 873 amino acid kinase repressor of Ras (KSR-1) was amplified to add a flag tag and the resultant product was cloned into the pTARGET™ Mammalian Expression Vector. The protein was expressed in COS7 cells and used for subcellular fractionation studies. (1434)

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