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PLos ONE 12, e0174916. In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection 2017

Nishimori, A. Konnai, S. Okagawa, T., Maekawa, N., Ikebuchi, R.,  Goto, S., Sajiki, Y.,  Suzuki, Y., Kohara, J. Ogasawara, S., Kato, Y.,  Murata, S. and Ohashi, K.

Notes: cDNA synthesized from RNA obtained from a rat hybridoma that produced a monoclonal antibody specific to bovine PD-1 was amplified and cloned into the pGEMT®-T Easy Vector. Additionally the Wizard® Genomic DNA Purification Kit was used to purify DNA from peripheral blood mononuclear cells (PBMCs) obtained from genomic DNA was extracted from antibody-inoculated cattle. (4771)

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PLos ONE . TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element 2017

Spaller, T., Groth M., Glöckner, G and Winckle, T.

Notes: After enrichment by LAM-PCR, products were gel purified and cloned into pGEM-T vector for manual sequencing reactions. Additionally, products produced from RT-PCR on circularized RNA were analyzed on agarose gels, cloned into pGEM-T vectors and sequenced. (4768)

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Neurobiol. Dis. 85, 35-48. Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease. 2016

Zizioli, D., Tiso, N., Guglielmi, A., Saraceno, C., Busolin, G., Giuliani, R., Khatri, D., Monti, E., Borsani, G., Argenton, F. and Finazzi, D.

Notes: To express the pank2 gene product, expression vectors were transfected into COS7, HeLa and SH-SY5Y cells using ViaFect™ Transfection Reagent (105 cells per chamber of glass slides; 1µg expression vector DNA: 3µl ViaFect™ Reagent). The cDNA clones used in this study were amplified by RT-PCR using ImProm-II™ Reverse Transcriptase for cDNA synthesis and Pfu DNA Polymerase for PCR. PCR products were cloned into the pGEM®-T Easy Vector. (4663)

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Mem Inst Oswaldo Cruz. 111, 287–93. Zika virus damages the human placental barrier and presents marked fetal neurotropism 2016

Noronha, Ld., Zanluca, C., Azevedo, M.L., Luz, K.G. and Santos, C.N.

Notes: The authors demonstrate evidence of transplacental transmission of Zika virus through detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestational. To confirm the identity of the flavivirus in the IHC assays, the corresponding FFPE tissue block was punched with a hollow needle, and tissue cores 3 mm in width were removed for molecular studies. Total RNA was extracted from these cores using the ReliaPrepTM FFPE total RNA Miniprep System according to the manufacturer’s recommendations. RNA was eluted in 50μL of elution buffer, and 5 μL of the extracted RNA was amplified by real-time RT-PCR using two primer/probe sets specific for ZIKV (Lanciotti et al. 2008). Real-time assays were performed using the GoTaq Probe 1-Step RTqPCR System. (4719)

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J. Biotechnol. 166, 42–50. Molecular characterization of the first transgenic common bean immune to the Bean golden mosaic virus 2013

Aragão, F.J., Noqueira, E.O. Tinoco, M.F. and Faria, J.C.

Notes: PCR products from a tertiary TAIL-PCR were separated by agarose gel electrophoresis and purified using the Wizard® SV Gel and PCR Clean-Up Kit. Purified fragments were cloned into pGEM®-T Easy Vectors, and clones were sequenced. (4547)

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Proc. Natl. Acad. Sci. USA 109, 2654–9. A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice 2012

Ding, J., Lu Q, Ouyang, Y., Mao, H., Zhang, P., Yao, J., Xu, C. Li, X., Xiao, J. and Zhang, Q.

Notes: Genomic DNA was isolated from rice plants and bisulfite treated. Target regions were specifically amplified using bisulfite primers, and the amplified products cloned into the pGEM®-T vector prior to large scale bisulfite sequencing. Sequencing analysis was performed using the Kismeth tool. pGEM®-T was also used as the vector for fragments amplified from cDNA clones of transcripts of interest prior to generating sense and antisense RNA probes for RNA in situ hybridization experiments. Apoptosis of tissue from anthers was assessed using the DeadEnd Fluorometric TUNEL System. (4558)

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PLos ONE 7, e47892. Epigenetic disruption of the PIWI pathway in human spermatogenic disorders. 2012

Heyn, H., Ferreira, H.J., Bassas, L., Bonache, S., Sayols, S., Sandoval, J., Esteller, M. and Larriba, S.

Notes: The authors used microarray analysis, bisulfite sequencing and pyrosequencing to examine a possible link between aberrant DNA methylation and abnormal human spermatogenesis and male infertility. They identified almost 600 genes that were differentially methylated in testis tissue of men with secretory male infertility. Genomic DNA used in the microarray analysis was extracted from testicular biopsies using the Wizard® Genomic DNA Purification Kit. For the bisulfite sequencing experiments, genomic DNA was bisulfite-modified, amplified, cloned using the pGEM®-T Easy Vector System, then sequenced. (4257)

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Veterinary Microbiology 160(3-4), 463–467. Molecular detection of murine noroviruses in laboratory and wild mice. 2012

Farkas, T., Fey, B., Keller, G., Martella, V. and Egyed, L.

Notes: Mice RNA samples were converted to cDNA using an oligo-dT primer with the Reverse Transcription System, ethanol precipitated, vacuum dried and transferred to another lab. There they were reconstituted in 20μl of molecular biology grade water.

Detection of caliciviruses in the wild mice samples was attempted using generic calicivirus primers targeting sequences encoding conserved amino acid motifs in the RNA-dependent RNA polymerase (RdRp) region of ORF1. Two microliters of cDNA was used in 25μl PCR reactions using the GoTaq® Green Master Mix. Laboratory mouse RNA samples were tested only with MNV-specific primers in the AccessQuick™ RT-PCR System using 2μl RNA as template.

PCR products were cloned into pGEM-T® Vector and sequenced using M13 forward and reverse primers on an ABI PRISM® 3730 DNA Analyzer. (4330)

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J. Biol. Chem. 287, 22969-22987. Pink1 kinase and its membrane potential (Deltaψ)-dependent cleavage product both localize to outer mitochondrial membrane by unique targeting mode. 2012

Becker, D., Richter, J., Tocilescu, M,A., Przedborski, S., and Voos, W.

Notes: The authors of this paper studied the targeting mode of the Parkinson disease-associated kinase Pink1. They constructed a number of truncation and deletion mutants in the pGEM®-4Z Vector, and verified the identity of these clones using next-generation sequencing. The authors then expressed radiolabeled versions of the various Pink1 constructs using the TNT® Coupled Rabbit Reticulocyte Lysate System. These labeled proteins were used in mitochondrial localization studies.   (4563)

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J. Virol. 86, 10999–11012. Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces. 2012

Wu, Z., Ren, X., Yang, L., Hu, Y., Yang, J., He, G., Zhang, J., Dong, J., Sun, L., Du, J., Liu, L., Xue, Y., Wang, J., Yang, F., Zhang, S. and Jin, Q.

Notes: Swab samples from 11 species of Chinese bats were vortexed in maintenance medium, filtered through a 0.45µm pore filter, centrifuged and resuspended. Any nonencapsidated (naked) nucleic acid was digested in a cocktail of enzymes including 20U of RNase ONE™ Ribonuclease prior to DNA and RNA purification. Conserved regions of the RNA-dependent RNA polymerase gene of astroviruses were reverse transcribed, amplified and cloned into the pGEM®-T Easy Vector for sequencing. (4552)

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J. Biol. Chem. 286, 37196–206. 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-myc nuclear localization and binding to the e-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes. 2011

Ning, B., Liu., G., Liu, Y., Su, X., Anderson, G.J., Zheng, X., Chang, Y., Guo, M., Liu, Y., Zhao, Y. and Nie, G.

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

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Appl. Environ. Microbiol. 77, 2113–21. General suppression of Escherichia coli O157:H7 in sand-based dairy livestock bedding. 2011

Westphal, A., Williams, M.L., Baysal-Gurel, F., LeJeune, J.T. and McSpadden Gardener, B.B.

Notes: The authors investigated the suppression of E. coli O157:H7 in sand-based livestock bedding and hypothesized that suppression of E. coli O157:H7 growth was mediated by an environmentally stable population of pathogen-suppressing bacteria. These bacteria were identified by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA gene sequences isolated from used bedding followed by cloning and sequencing of the most abundant terminal restriction fragments. Amplifications were performed using the GoTaq® Flexi DNA Polymerase, then PCR products were cloned into the pGEM®-T Easy Vector. The PureYield™ Plasmid Miniprep System was used to purify plasmids for sequencing. (4165)

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Nucl. Acids Res. Dec 8, Epub ahead of print. Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts. 2011

Zhelyazkhova, P., Hammani, K., Rojas, M., Voelker, R., Vargas-Suárez, M., Börner, T., and Barkan, A.

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

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Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

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J. Biol. Chem. 286, 19478–19488. Thrombomodulin is silenced in malignant mesothelioma by a poly(ADP-ribose) polymerase-1-mediated epigenetic mechanism. 2011

Nocchi, L., Tomasetti, M., Amati, M., Neuzil, J., Santarelli, L. and Saccucci, F.

Notes: Thrombomodulin (TM) expression was examined by isolating genomic DNA from biopsies of human malignant mesothelioma and normal mesothelial tissue, and cultured cell lines with or without PARP1 silencing treated with 5-aza-2´-deoxycytidine and trichostatin alone or in combination and then subjected to biosulfide modification. To analyze methylation of TM, a CpG island in the promoter, 5´ UTR and an exon region containing 44 CpG dinucleotides were PCR amplified, cloned into the pGEM®-T Easy Vector, transformed and positive clones selected using IPTG/X-Gal and analyzed by PCR. Colonies were cultured, the plasmids isolated using the Wizard® Plus SV Minipreps DNA Purification System then 10 clones
from each sample type were sequenced. (4132)

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Appl. Environ. Microbiol. 75, 2275–83. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by gacA. 2009

Shi, X.Y., Dumenyo, C.K., Hernandez-Martinez, R., Azad, H. and Cooksey, D.A.

Notes: To gain a better understanding of how Xylella fastidiosa causes diseases in grapes, the authors mutated conserved regulatory genes, including gacA, that affect expression of virulence-related factors in other species. The relative expression levels of gacA in wildtype and mutated strains were examined using RT-PCR. The authors also identified and quantified a number of genes that were regulated by GacA by microarray analysis. Microarray results were confirmed using RT-PCR. RT-PCR was performed using the AccessQuick™ RT-PCR System. (4052)

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

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

Notes: The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER® Vector and amino acid substitutions were introduced using the Altered Sites® II in vitro Mutagenesis System. The pGEM®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase® Reporter Assay System. (4023)

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

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

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

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

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

Notes: The authors investigated gene transcription within periplasmic flagella of Borrelia burgdorferi, which are composed of a basal body, hook and filament, to determine if hook formation influences flagellin gene expression. They used insertion mutagenesis to construct strains with mutated versions of the hook structural gene flgE that were disrupted by a kanamycin-resistance cassette. The flgE gene and antibiotic-resistance cassette were amplified by PCR and cloned into the pGEM®-T Vector. To assess the effect of flgE disruption on the transcription of filament proteins FlaA and FlaB, quantitative RT-PCR was performed; enolase was used as an internal control. Negative controls without the reverse transcriptase were included for each sample. (3885)

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J. Virol. doi:10.1128/JVI.01509-08, Epub (ahead of print). Classical swine fever virus can remain virulent after specific elimination of the interferon regulatory factor 3 degrading function of Npro. 2008

Ruggli, N., Summerfield, A., Fiebach, A.R., Guzylack-Piriou, L., Bauhofer, O., Lamm, C.G., Waltersperger, S., Matsuno, K., Liu, L., Gerber, M., Choi, K.H., Hofmann, M.A., Sakoda, Y., Tratschin, J.D.

Notes: These authors studied the effect of specific amino acid substitutions in the Npro gene of swine fever virus. The Npro gene encodes a non-strucutral protein that prevents interferon production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3). Npro also has an autoprotease function. Deletion of the entire Npro region attenuates virulence. In this study, the authors showed that degradation of IRF3 and autoprotease activity are independent, structurally overlapping functions. In particular, they investigated the effect of specific amino acid substitutions that eliminated IRF3 interaction and degradation, but did not affect autoprotease activity. They showed that removal of IRF3 degradation activity of Npro had only minimal effect on virulence in swine. The pGEM-T Vector was used to clone the amplified Npro gene, and the CheckMate™ Flexi Vector Mammalian Two-Hybrid System was used for protein interaction studies. (3944)

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Antimicrob. Agents Chemother. 52, 1812–9. Enhanced resistance to bacterial infection in protegrin-1 transgenic mice. 2008

Cheung, Q.C., Turner, P.V., Song, C., Wu, D., Cai, H.Y., MacInnes, J.I. and Li, J.

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. (3896)

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Microbiology 154, 139–47. Involvement of BmoR and BmoG in n-alkane metabolism in 'Pseudomonas butanovora'. 2008

Kurth, E.G., Doughty, D.M., Bottomley, P.J., Arp, D.J. and Sayavedra-Soto, L.A.

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. (3893)

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Nucl. Acids Res. 36, 2107–2722. PDZ domain-mediated dimerization and homeodomain-directed specificity are required for high-affinity DNA binding by SATB1. 2008

Purbey, P.K., Singh, S., Kumar, P.P., Mehta, S., Ganesh, K.N., Mitra, D. and Galande, S.

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. (3982)

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Clin. Vaccine Immunol. 15, 418–424. Sequential analysis of Anaplasma phagocytophilum msp2 transcription in murine and equine models of human. 2008

Scorpio, D.G., Leutenegger, C., Berger, J., Barat, N., Madigan, J.E. and Dumler, J.S.

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. (3975)

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