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Mol. Cell. Biol. 25, 5763-76. Generation and characterization of Rac3 knockout mice. 2005

Corbetta, S., Gualdoni, S., Albertinazzi, C., Paris, S., Croci, L., Giacomo Consalez, G., and de Curtis, I.

Notes: GoTaq® DNA Polymerase was used in an RT-PCR. Aliquots of RNA were transcribed into cDNA and amplified with GoTaq® DNA Polymerase using primers specific for Rac1. (3351)

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Vet. Parasitol. 133, 283-287. Genotyping of Giardia intestinalis from domestic and wild animals in Japan using glutamete dehydrogenase gene sequencing. 2005

Itagaki, T., Kinoshita, S., Aoki, M., Itoh, M., Saeki, H., Sato, N., Uetsuki, J., Izumiyama, S., Yagita, K., and Endo, T.

Notes: GoTaq® DNA Polymerase was used in PCR genotyping of Giardia intestinalis. Primers were designed around a 177 bp sequence of the glutamete dehydrogenase gene (gdh). Typing was based on previously reported assemblages of gdh from cats, dogs, cows and monkeys. PCR was performed in a total reaction volume of 25μl using 1.25 units of GoTaq® DNA Polymerase.
(3364)

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Mol. Pharmacol. 67, 375–382. Helix I of β-arrestin is involved in postendocytic trafficking but is not required for membrane translocation, receptor binding, and internalization. 2005

Dinh, D.T., Qian, H., Seeber, R., Lim, E., Pfleger, K., Eidne, K.A. and Thomas, W.G.

Notes: Type 1 angiotensin II receptor-Renilla luciferase (AT1R-Rluc), and β-arrestin1 and 2 GFP  fusion constructs (βarr1-GFP and βarr2-GFP) were created for BRET protein interaction assays. Combinations of AT1R-Rluc and β-arrestin-GFP constructs were transfected into COS-7 cells. The COS-7 cell cultures were then activated with 100nM angiotensin II in the presence of 60μM EnduRen™ Live Cell Substrate, and BRET fluorescence readings were taken at 475 and 515 nm over a 1 hour period. The authors also describe analysis of helix I mutants of β-arrestin1 and β-arrestin2 in similar β-arrestin-GFP construct BRET studies.  Data are displayed as a ratio of fluorescence readings with both constructs compared to fluorescence from the AT1R-Rluc construct alone.  (3256)

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Nucl. Acids Res. 33, e158. Improved methods for the generation of human gene knockout and knockin cell lines. 2005

Topaloglu, O., Hurley, P.J., Yildirim, O., Civin, C.I. and Bunz, F.

Notes: To examine both the efficiency of gene targeting constructs and the ability to knockin or knockout a gene, recombinant associated adenovirus (rAAV) constructs for p53 and CTNNB1 were created. Either HCT116 or DLD1 human colorectal cancer cell lines were infected with the rAAV and selected for resistance to Geneticin®. After the drug-resistant colonies were grown for 3–4 weeks, the genomic DNA from each clone was isolated using the Wizard® SV 96 Genomic DNA Purification System and locus-specific integration assessed by PCR. (3556)

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Croat. Med. J. 46, 563–577. Improving efficiency of a small forensic DNA laboratory: validation of robotic assays and evaluation of microcapillary array device. 2005

Crouse, C.A., Yeung, S., Greenspoon, S., McGuckian, A., Sikorsky, J., Ban, J. and Mathies, R.

Notes: The authors validated the DNA IQ™ System for manual and automated DNA purification from blood, tissue, bone, hair, epithelial cells and mixed stains such as semen. Automated DNA extraction was performed using the Beckman Coulter Biomek® 2000 workstation. Purified DNA (0.5–1.0ng) was then amplified using the PowerPlex® 16 BIO System and a 6% PAGE PLUS™ gel. Automated DNA purification and use of a single-amplification STR system greatly increased sample throughput. (3645)

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Toxicol. Sci. 85, 727-734. Inhibitory effects of vitamin A on TCDD-induced cytochrome P-450 1A1 enzyme activity and expression. 2005

Yang, Y.M., Huang, D.Y., Liu, G.F., Zhong, J.C., Du, K., Li, Y.F. and Song. X.H.

Notes: The ability of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to upregulate cytochrome P450 CYP1A1 levels in mouse liver was examined. Total mouse liver RNA was reverse transcribed using the Reverse Transcription System, and the resulting CYP1A1 cDNA was quantitated using real-time PCR. CYP1A1 protein levels were quantitated by Western blot using an anti-CYP1A1 antibody, the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate secondary antibody and the Western Blue® Stabilized Substrate for Alkaline Phosphatase.
(3443)

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Clin. Can. Res. 11, 8699-8705. Methylation of the DPYD promoter: an alternative mechanism for dihydropyrimidine dehydrogenase deficiency in cancer patients. 2005

Ezzeldin, H.H., Lee, A.M., Mattison, L.K., and Diasio, R.B.

Notes: To examine the methylation status of the DPYD promoter, genomic DNA was extracted from an RKO cell line and from peripheral blood mononuclear cells using the Wizard® SV Genomic DNA Purification System. The isolated DNA was treated by sodium bisulfate modification followed by amplification and sequencing, or by DHPLC to determine CpG island methylation state. (3587)

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Infect. Immun. 73, 2611-2620. Molecular cloning and characterization of three beta-defensins from canine testes. 2005

Sang, Y., Ortega, M.T., Blecha, F., Prakash, O. and Melgarejo, T.

Notes: The investigators cloned three β-defensins, which are antimicrobial peptides, from canine testes. A canine expressed sequence tag (EST) was identified based on similarity to human β-defensins. Full-length cDNAs were obtained using 5´- and 3´RACE, then amplified by PCR and cloned into the pGEM®-T Easy Vector. cDNA sequences were confirmed using the SP6 and T7 Promoter Primers. The tissue-specific expression of canine β-defensins (cBDs) was characterized using the AccessQuick™ RT-PCR System to amplify β-defensin RNA from a variety of tissues. RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. The identity of the RT-PCR products was confirmed by electrophoresis, transfer to nylon membranes and hybridization to probes derived from sequence-confirmed β-defensin clones; the probes were synthesized using the Prime-a-Gene® Labeling System. To localize expression of the three β-defensin isoforms in canine testes, in situ hybridization (ISH) was performed. The 3´-RACE products were cloned into the pGEM®-T Vector, which was then linearized and treated with exonuclease III to delete an approximately 80-bp region shared by the three cBD isoforms. The resulting product was used to synthesize sense and antisense ISH probes. (3453)

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Intl. J. Parasitol. 35, 1071-1078. Molecular data suggest that microsporidian parasites in freshwater snails are diverse. 2005

McClymont, H.E., Dunn, A.M., Terry, R.S., Rollinson, D., Littlewood, D.T., and Smith, J.E.

Notes: Researchers tested several snail samples from various species for the presence of microsporidia infection. PCR tests were performed with GoTaq® DNA Polymerase and primers specific to microsporidia 16S DNA sequences. Isolated snail/microsporidia DNA was amplified in 25µl reactions with 0.625 units of GoTaq® DNA Polymerase. (3378)

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J. Gen. Virol. 86, 623-630. Mutation of chicken anemia virus VP2 differentially affects serine/threonine and tyrosine protein phosphatase activities. 2005

Peters, M.A., Jackson, D.A., Crabb, B.S. and Browning, G.F.

Notes: This study investigated the role of the dual-specificity protein phosphatase, Viral Protein 2 (VP2) from chicken anemia virus in virus-induced immunosuppression. Mutations were introduced into the VP2 sequence by overlap-expression PCR. Serine/Threonine phosphatase activity of VP2 mutants was investigated using the Serine/Threonine Phosphatase Assay System. (3525)

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Infect. Immun. 73, 7064-7068. Mycobacterial lipomannan induces matrix metalloproteinase-9 expression in human macrophagic cells through a Toll-like receptor 1 (TLR1)/TLR2- and CD14-dependent mechanism. 2005

Elass, E., Aubry, L., Masson, M., Denys, A., Guérardel, Y., Maes, E., Legrand, D., Mazurier, J., and Kremer, L.

Notes: M-MLV Reverse Transcriptase was used to make cDNA from differentiated THP-1 cell total RNA. For these reverse transcription experiments the authors used 5 µg of total RNA, oligo dT and RNasin®. GoTaq® DNA Polymerase was used to amplify the cDNA from the reverse transcription reactions. PCR products were separated by gel electrophoresis and analyzed by ethidium bromide staining and band densitometry. (3357)

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J. Biol. Chem. 280, 1376-1383. PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum. 2005

van Lith, M., Hartigan, N., Hatch, J. and Benham, A.M.

Notes: The tissue-specific patterns of expression of the novel protein disulfide isomerase-like protein of the testis (PDILT) was characterized using the AccessQuick™ RT-PCR System. RNAs isolated from various mouse tissues were amplified to reveal the testis-specific expression. An in vitro PDILT transcript generated using the Riboprobe® System-T7 was used as a positive control. Actin mRNA was also amplified to demonstrate equal RNA input. (3433)

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J. Virol. 78, 9936-9946. Poly(ADP-Ribose) polymerase 1 binds to Kaposi's sarcoma-associated Herpesvirus (KSHV) terminal repeat sequence and modulates KSHV replication in latency. 2005

Ohsaki, E., Ueda, K., Sakakibara, S., Do, E., Yada, K. and Yamanishi, K.

Notes: Three terminal repeat sequences (TR) from Kaposi's sarcoma-associated herpesvirus (KSHV) were PCR amplified and then purified using the Wizard® PCR Preps DNA Purification System. The PCR products were 386, 424, and 307 base pairs in size.  These TR products were used in PARP1-DNA ELISA assays.  The authors also used the Wizard® SV Genomic DNA Purification System to isolate DNA from a BC3 cell line infected with KSHV. The isolated DNA was used in real-time PCR to assess KSHV copy number in cells. (3232)

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Toxicol. Sci. 85, 632-638. Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu. 2005

van de Bovenkamp, M., Groothuis, G.M.M., Draaisma, A.L., Merema, M.T., Bezuijen, J.I., van Gils, M.J., Meijer, D.K.F., Friedman, S.L. and Olinga, P.

Notes: Rat liver slices were used to study hepatic stellate cell (HSC) activation and fibrogenesis. The expression of markers of HSC activation and fibrogenesis were analyzed by real-time PCR. To quantitate mRNA levels, total rat liver RNA was reverse transcribed using the Reverse Transcription System, then amplified in the presence of SYBR® Green. (3430)

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J. Clin. Microbiol. 43, 6027-6031. Presence of Rickettsia conorii subsp. israelensis, the causative agent of Israeli spotted fever, in Sicily, Italy, ascertained in a retrospective study. 2005

Giammanco, G.M., Vitale, G., Mansueto, S., Capra, G., Caleca, M.P. and Ammatuna, P.

Notes: The authors infected Vero cells with the rickettsial agent, then recovered bacterial DNA from the infected cells using the Wizard® Plus SV Minipreps DNA Purification System, and amplified this DNA by PCR. (3541)

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RNA 11, 1571-8. Rat1p and Rai1p function with nuclear exosome in the processing and degradation of rRNA precursors. 2005

Fang, F., Phillips, S. and Butler, J.S.

Notes: GoTaq® DNA Polymerase was used in a RT-PCR. The 50μl PCR reactions contained 0.25μl cDNA, 12.5pmol primer, 0.025mM dNTPs, 2.5mM MgCl2 and 0.25μl of GoTaq® DNA Polymerase (5U/μl). PCR was performed using the following conditions: 94°C for 3 minutes followed by 20 cycles (94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds), followed by 72°C for 7 minutes and held at 4°C. Products were visualized on an agarose gel. (3352)

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RNA 11, 1571–8. Rat1p and Rai1p function with the nuclear exosome in the processing and degradation of rRNA precursors. 2005

Fang, F., Phillips, S. and Butler, J.S.

Notes: To examine the effect of the exoribonuclease Rat1p on processing of ribosomal RNAs (rRNA), total RNA was isolated from several yeast strains with altered RAT1 expression. One microgram of DNase-treated total RNA was reverse transcribed in a 20µl reaction. Subsequently, 0.25µl of cDNA product was amplified using 0.25µl of GoTaq® DNA polymerase in a total volume of 50µl. The RT-PCR products were separated on 2% agarose gels and stained using ethidium bromide. (3330)

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Am. J. Med. Genet. 138A, 99–106. Screening for new MTHFR polymorphisms and NTD risk. 2005

O'Leary, V.B., Mills, J.L., Parle-McDermott, A., Pangilinan, F., Molloy, A.M., Cox, C., Weiler, A., Conley, M., Kirke, P.N., Scott, J.M., Brody, L.C. and Birth Defects Research Group.

Notes: The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) has at least one polymorphism that is a neural tube defect (NTD) risk factor within the Irish population. To survey for common variations in MTHFR, genomic DNA was extracted from blood, and exons 1–11 of MTHFR were amplified and sequenced with Big Dye® Terminator mix. The Wizard® MagneSil® Sequencing Reaction Clean-Up System was used to purify the sequencing reactions prior to analysis on an ABI PRISM® 377 DNA sequencer. (3444)

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J. Biol. Chem. 280, 36802–8. Structural and genetic analyses reveal a key role in prophage excision for the TorI response regulator inhibitor. 2005

Elantak, L., Ansaldi, M., Guerlesquin, F., Mejean, V. and Morelli, X.

Notes: To test the activity of torI as a prophage excisionase, the chloramphenical aceytltransferase gene was introduced into a non-coding region of the prophage KplE1 in a strain of E. coli containing a torI-encoding plasmid. Expression of torI was induced by IPTG and the cells plated on either chloramphenical or ampicillin. GoTaq® DNA polymerase was used in a PCR test to confirm prophage DNA excision from randomly chosen ampicillin-resistant colonies. (3329)

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J. Biol. Chem. 280, 36802-36808. Structural and genetic analyses reveal a key role in prophage excision for the torl response regulator inhibitor. 2005

ElAntak, L., Ansaldi, M., Guerlesquin, F., Méjean, V. and Morelli, X.

Notes: GoTaq® Polymerase was used in this study investigating the role of TorI (Tor inhibition protein) in prophage DNA excision. PCR products were amplified from E. coli strains carrying a plasmid-encoded torI gene, and were analyzed by gel electrophoresis. (3350)

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J. Bacteriol. 187, 3762-3778. Sulfur amino acid metabolism and its control in Lactococcus lactis IL1403. 2005

Sperandio, B., Polard, P., Ehrlich, D.S., Renault, P. and Guédon, E.

Notes: To study the transcription levels of several genes involved in cysteine and methionine metabolism, Lactococcus lactis strains deficient in these genes were created by double-crossover homologous integration. To do so, PCR was performed to amplify sequences upstream and downstream of the target gene, and the PCR products were ligated and cloned into the pGEM®-T Easy Vector. The resulting vectors were fused to pGhost9, a vector designed for homologous recombination in L. lactis. Once the gene deletions were verified, quantitative real-time RT-PCR was performed to quantitate expression levels of a variety of genes involved in Cys and Met metabolism. (3461)

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Nucl. Acids Res. 33, e41. Targeted ‘knockdown’ of spliceosome function in mammalian cells. 2005

Matter, N. and König, H.

Notes: PCR was performed in 50μl GoTaq® Reaction Buffer containing 5μl of diluted (1:10) reverse transcriptase reactions, 250mM each dNTP, 10 pmol primers and 2.5 U GoTaq® DNA Polymerase. A total of 28 cycles were performed for amplification of P120 minigene transcripts, and 31 cycles were performed for endogenous P120 and ADPRT transcripts. Each cycle consisted of 95°C for 1 min, 59°C (P120) or 58°C (ADPRT) for 1 min and 72°C for 1.5 min. (3353)

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Nucl. Acids Res. 33(4), e41. Targeted 'knockdown' of spliceosome function in mammalian cells. 2005

Matter, N. and Konig, H.

Notes: To examine why there are two splicing systems present in multicellular organisms, the authors used morpholino oligomers complementary to the branch-site recognition elements of U2 or U12 small nuclear RNA to specifically suppress the splicing function in EL4 and Jurkat cells. After transfection with the morpholino oligos, the cells were harvested, and total RNA was isolated followed by DNase-treatment. In a 20µl reverse transcription reaction, 0.8µg total RNA was reverse transcribed with 100ng oligo(dT)15 primer and 200 units of M-MLV Reverse Transcriptase, RNase H Minus. PCR was then performed using 5µl of the diluted (1:10) reverse transcriptase reaction with 2.5 units GoTaq® DNA polymerase in a 50µl reaction for 28–31 cycles. (3278)

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Biol. Reprod. 73, 745–51. The translationally controlled tumor protein is a novel calcium binding protein of the human placenta and regulates calcium handling in trophoblast cells. 2005

Arcuri, F., Papa, S., Meini, A., Carducci, A., Romagnoli, R., Bianchi, L., Riparbelli, M.G., Sanchez, J.C., Palmi, M., Tosi, P., and Cintorino, M.

Notes: These authors examined the role of translationally controlled tumor protein (TPT1) in human placental calcium transport. To test this, expression of TPT1 in the HTR-8/SVneo human trophoblast cell line was silenced by RNA interference. The TPT1 sequence corresponding to nucleotides 317 to 337 was targeted and the corresponding mouse TPT1 sequence was used as a control. The TPT1 sequence oligos for a hairpin siRNA were cloned into the psiSTRIKE™ Puromycin Vector and grown in JM109 cells. The purified plasmids were then transfected into HTR-8/SVneo cells at 60% to 80% confluence with 2µg human or control mouse shRNA vector. The cells were selected for puromycin resistance for two weeks and changes in TPT1 levels were determined by Western blot analysis. (3296)

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Proc. Natl. Acad. Sci. USA 102, 7701-7706. The two-component signal transduction system RR06/HK06 regulates expression of cbpA in Streptococcus pneumoniae. 2005

Standish, A.J., Stroeher, U.H., and Paton, J.C.

Notes: In this study, the binding of the response regulator RR06 to the promoter region of cbpA was investigated. DNA encoding the rr06 gene was cloned into the pGEM®-T Easy vector and transformed into E. coli strain DH5α. Cell lysates were prepared and incubated with a labeled cbpA promoter fragment, and binding was evaluated in an electrophoretic mobility shift assay (EMSA). To further investigate the RR06/cbpA interaction, the cbpA promoter region, or a control rRNA sequence, was biotin labeled and attached to Streptavidin MagneSphere® Paramagnetic Particles. The beads were incubated with E. coli lysates expressing RR06, or with control lysates. After washing, bound proteins were eluted by boiling. Eluted samples were then probed with specific anti-RR06 antisera. (3397)

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