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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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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. Biol. Chem. 283, 21579–87. ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action. 2008

Betzenhauser, M.J., Wagner, L.E. 2nd, Iwai, M., Michikawa, T., Mikoshiba, K. and Yule, D.I.

Notes: The authors examined the different ATP sensitivities of inositol (1,4,5)-triphosphate receptor (InsP3R) isoforms InsP3R1, InsP3R2 and InsP3R3. To compare the ATP-binding properties of InsP3R2 and InsP3R3, nucleotide sequences encompassing the ATP-binding domains were amplified by PCR and cloned into the pFN2A (GST) Flexi® Vector. The ATP-binding sites were expressed as glutathione-S-transferase (GST) fusion proteins in BL21(DE3)pLysS cells. Fusion proteins were purified, and the GST tag removed by cleavage with tobacco etch virus (TEV) protease. Purified proteins were then used in a fluorescent ATP-binding assay. (3901)

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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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Biol. Reprod. 79, 594–7. Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially? 2008

Bermejo-Alvarez, P., Rizos, D., Rath, D., Lonergan, P. and Gutiérrez-Adán, A.

Notes: To determine whether oocytes are able to select X-bearing or Y-bearing spermatozoa, the authors performed in vitro fertilization of bovine oocytes with X-sorted semen, Y-sorted semen, a mixture of X- and Y-sorted semen, and unsorted semen. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome-specific target for gender assignment and a bovine-specific satellite sequence as a control. PCRs were performed using GoTaq® Flexi DNA Polymerase (1 unit per 25µl reaction), and amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. (3881)

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Appl. Environ. Microbiol. 74, 312-318. Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitrosococcus oceani ATCC 19707. 2008

El Sheikh, A.F., Poret-Peterson, A.T. and Klotz, M.G.

Notes: These authors investigated the amo operon of the marine ammonia oxidizer Nitrosococcus oceani. The bacteria were grown at 30°C for 3 weeks in 200-400ml batch cultures in artificial seawater in the dark without shaking. Genomic DNA was isolated from cells in stationary phase using the Wizard® Genomic DNA Purification Kit. The isolated DNA was then used for PCR analysis. (3740)

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J. Biol. Chem. 283, 8218–28. Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway. 2008

Miao, Q., Sun, Y., Wei, T., Zhao, X., Zhao, K., Yan, L., Zhang, X., Shu, H. and Yang, F.

Notes: The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT® Coupled Transcription Translation System and [35S] methionine. (3889)

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Genetics 178, 1415–29. Comparative genetics of sex determination: masculinizing mutations in Caenorhabditis briggsae. 2008

Kelleher, D.F., de Carvalho, C.E., Doty, A.V., Layton, M., Cheng, A.T., Mathies, L.D., Pilgrim, D. and Haag, E.S.

Notes: The authors characterized masculinizing mutations of the female-promoting tra genes in Caenorhabditis briggsae (Cb-tra). Using RT-PCR, the authors monitored the levels of full-length Cb-tra mRNA and a novel splice variant; actin mRNA was amplified as a control. RT-PCR was carried out using the AccessQuick™ RT-PCR System and RNA from 5–10 worms per 50µl reaction. (3892)

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Mol. Cancer Res. 6, 546–54. CXCL16 functions as a novel chemotactic factor for prostate cancer cells in vitro. 2008

Lu, Y., Wang, J., Xu, Y., Koch, A.E., Cai, Z., Chen, X., Galson, D.L., Taichman, R.S. and Zhang, J.

Notes: The authors sought to detect expression of the chemokine CXCL16 and its receptor, CXCR6, in prostate cancer cell lines and in benign and malignant prostate cancer tissues. The Access RT-PCR System was used to amplify and detect CXCL16 and CXCR6 mRNA in these cells and tissues. Each RT-PCR contained 1µg of total RNA, and amplifications were carried out for 35 cycles. Amplified products were detected on a 1.5% ethidium bromide-stained agarose gel. (3836)

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Plant Physiol. 146, 1469–81. Deregulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant. 2008

Covshoff, S., Majeran, W., Liu, P., Kolkman, J.M., van Wijk, K.J. and Brutnell, T.P.

Notes: The authors identified the maize homolog of hcf136 (Zmhcf136), a gene involved in photosynthesis, and used an RNA blot to determine if ZmHcf136 transcripts accumulate preferentially in mesophyll cells. DNA probes for Zmhcf136 and several cell-specific markers were generated by PCR using GoTaq® Green Master Mix, gel purified and radiolabeled prior to use in the RNA blots. To examine differences in protein accumulation and localization in wildtype and hcf136 mutants, proteins from subcellular fractions were subjected to two-dimensional gel electrophoresis, and spots of interest were excised, digested with Sequencing Grade Modified Trypsin, then analyzed by electrospray ionization-tandem mass spectrometry. (3883)

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Forensic Sci. Int. Genet. 3, 14–21. Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA. 2008

Krenke, B.E., Nassif, N., Sprecher, C.J., Knox, C., Schwandt, M. and Storts, D.R.

Notes: The authors describe the developmental validation of the Plexor® HY System, a quantitative PCR assay that simultaneously quantifies total human and male DNA. Validation studies examined: (1) human specificity, (2) sensitivity, (3) quantitation of degraded DNA, (4) impact of inhibitors, (5) male/female mixture and Y-assay male specificity, (6) reproducibility and concordance and (7) population studies. (3969)

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Stem Cells 26, 1841-1849. Embryonic Stem Cells as a Platform for Analyzing Neural Gene Transcription 2008

Zhang, X., Horrell, S.A., Delaney, D., Gottlieb, D.I.

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 controlling 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. (3918)

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J. Clin. Microbiol. 46, 1741–1746. High-throughput genotyping of Salmonella enterica serovar Typhi allowing geographical assignment of haplotypes and pathotypes within an urban District of Jakarta, Indonesia. 2008

Baker, S., Holt, K., van de Vosse, E., Roumagnac, P., Whitehead, S., King, E., Ewels, P., Keniry, A., Weill, F.X., Lightfoot, D., van Dissel, J.T., Sanderson, K.E., Farrar, J., Achtman, M., Deloukas, P. and Dougan, G.

Notes: The authors examined strains of Salmonella enterica serovar Typhi isolated from typhoid cases originating in or around Indonesia or from travelers returning from Indonesia to examine if serovar Typhi from this area has a greater level of genetic diversity compared to other countries. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit, diluted to 4 ng/µl and used in locus-specific PCR genotyping. (3980)

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Mol. Med. Reports Atenas 1, 123–129. HPV infection in Brazilian oral squamous cell carcinoma patients and its correlation with clinicopathological outcomes 2008

Oliveira, L.R., Ribeiro-Silva, A., Ramalho, L.N.Z., Simões, A.L. and Zucoloto, S.

Notes: In this study of the frequency of human papilloma virus (HPV) in patients with oral squamous cell carcinoma (OSCC), the MagneSil® Genomic, Fixed Tissue System was used to isolate DNA from formalin-fixed paraffin-embedded tissue samples from primary tumors and matched samples. Five microliters of genomic DNA was amplified using PCR Master Mix and primers for both a 110 bp fragment of human ß-globin gene and HPV genotype. After PCR, the product was analyzed on an 8% nondenaturing polyacrylamide gel and stained with AgNO3. (3938)

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Cancer Res. 2007, 6146–54. Incorporation of an internal ribosome entry site-dependent mechanism in arsenic-induced GADD45 alpha expression. 2008

Chang, Q,. Bhatia, D., Zhang, Y., Meighan, T., Castranova, V., Shi, X. and Chen, F.

Notes: Trivalent arsenic (As3+) induces expression of growth arrest- and DNA damage-induced gene 45α (GADD45α), which interacts with intracellular signaling molecules involved in cell cycle regulation, apoptosis and immune response. To characterize GADD45α mRNA expression patterns, total RNA was isolated from untreated (growing) and As3+-treated (arrested) BEAS-2B cells, and GADD45α mRNA was amplified using the AccessQuick™ RT-PCR System. GADD45α mRNA levels were undetectable in growing cells but increased in a time-dependent manner in arrested cells. Reverse transcription was carried out at 45°C for 50 minutes, followed by 35 cycles of PCR. (3766)

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Cancer Res. 68, 4623–4630. Integrative genomics identifies RAB23 as an invasion mediator gene in diffuse-type gastric cancer. 2008

Hou, Q., Wu, Y.H., Grabsch, H., Zhu, Y., Leong, S.H., Ganesan, K., Cross, D., Tan, L.K., Tao, J., Gopalakrishnan, V., Tang, B.L., Kon, O.L. and Tan, P.

Notes: In this article, the researchers explored the role of RAB23 in gastric cancers. Twenty-four hours before transfection, AGS cells (gastric cancer cell line) that expressed RAB23 were seeded into a 24-well plate at a density of 1.8 × 105 cells/ml. To overexpress RAB23, a full-length RAB23 cDNA was cloned into the pCI-neo Mammalian Expression Vector and transfected into AGS cells. The effect of RAB23 overexpression on the cells was determined using a Matrigel invasion assay while protein expression levels were visualized with Western blotting. (3986)

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J. Cell Sci. 121, 504–13. Integrin alpha9 beta1 is a receptor for nerve growth factor and other neurotrophins. 2008

Staniszewska, I., Sariyer, I.K., Lecht, S., Brown, M.C., Walsh, E.M., Tuszynski, G.P., Safak, M., Lazarovici, P. and Marcinkiewicz, C.

Notes: The authors investigated the ability of α9β1 integrin to act as a neurotrophin receptor and affect cell signaling pathways. As part of the study, RT-PCR was used to detect the presence of other neurotrophin receptors in their model cell line, SW480. Reverse transcription was performed using the Reverse Transcription System and 1µg of total RNA isolated using the SV Total RNA Isolation System. The resulting cDNA (5µg) was amplified for 35 cycles (β-actin as a control) or 40 cycles (TrkA and p75NTR) using GoTaq® Green Master Mix. RT-PCR results were confirmed by Western blot analysis. (3884)

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Carcinogenesis 29, 1184-1191. Interaction of the cytochrome P4501A2, SULT1A1 and NAT gene polymorphisms with smoking and dietary mutagen intake in modification of the risk of pancreatic cancer. 2008

Suzuki, H., Morris, J.S., Li ,Y., Doll, M.A., Hein, D.W., Lium J., Jiao, L., Hassan, M.M., Day, R.S., Bondy, M.L., Abbruzzese, J.L., and Li, D.

Notes: In this study, the Maxwell® 16 Instrument was used to purify genomic DNA from blood samples. The extracted DNA was amplified by PCR for subsequent genotype analysis. (3902)

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J. Exp. Bot. 59, 2253–65. Interaction study of MADS-domain proteins in tomato. 2008

Leseberg, C.H., Eissler, C.L., Wang, X., Johns, M.A., Duvall, M.R. and Mao, L.

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. (3886)

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Proc. Natl. Acad. Sci. USA 105, 7141-7146. Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): A key to cellulase gene expression on lactose 2008

Fekete, E., Seiboth, B., Kubicek, C.P., Szentirmai, A., Karaffa, L.

Notes: To amplify yeast mutarotase, S. cerevisiae was used, and E. coli strain JM109 (Promega Cat.# L2001) was used for plasmid propagation. Fungal mycelia were harvested by filtration, washed, frozen and ground under liquid nitrogen. Genomic DNA was extracted using the Wizard Genomic DNA Purification System (Promega Cat.# A1120). RNA for hybridization and RT-PCR was extracted from mycelia using the SV Total RNA Isolation System (Promega Cat.# Z3101) and plasmid DNA isolated using the PureYield(TM) Plasmid Midiprep System (Cat.# A2492). (3919)

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J. Biol. Chem. May 6, Epub (ahead of print). Mechanism mediating the enhanced transcription of P2X3 receptor gene by calcitonin gene related peptide in trigeminal sensory neurons. 2008

Simonetti, M., Giniatullin, R., and Fabbretti, E.

Notes: These authors investigated the mechanism of action of the migraine mediator calcitonin gene-related peptide (CGRP), which is known to sensitize the P2X3 pain receptors to increase impulse flow to brain stem trigeminal nuclei. They showed that CAM KII inhibitors prevented CGRP-induced upregulation of P2X3 mRNA using real-time RT-PCR, and then confirmed that active CAM KII was involved in the signaling mechanism by staining with Anti-ACTIVE® CAM KII Antibodies. The immunoreactivity was upregulated by CGRP treatment of trigeminal neurons, and the distribution of the active CAM KII was localized to the membrane region. They then examined the mechanism of transcriptional activation of the P2X3 pain receptor genes and showed that the transcription factor CREB, which is known to be dependent on CAM KII for activation and nuclear localization, was involved. The authors also investigated the role of BDNF in the process, because CGRP is known to promote BDNF expression by trigeminal neurons, and because BDNF is thought to be involved in pain processing. They used the BDNF Emax Immunoassay System to measure BDNF levels in culture media after application of CGRP to neuronal cell cultures, and demonstrated that there was a fourfold increase in BDNF after CGRP exposure. They also showed that BDNF promoted CAMKII and CREB activation. (3873)

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Clin. Can. Res. 14, 5033–42. Midkine enhances soft-tissue sarcoma growth: a possible novel therapeutic target. 2008

Jin, Z., Lahat, G., Korchin, B., Nguyen, T., Zhu, Q.S., Wang, X., Lazar, A.J., Trent, J., Pollock, R.E. and Lev D.

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. (3895)

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Nucl. Acids Res. 36, 5391–404. miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes. 2008

Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.

Notes: To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control. (3894)

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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. Biomol. Scr. 13, 968–974. Mouse thymus targeted peptide isolated by in vivo phage display can inhibit bioactivity of thymus output in vivo. 2008

Yu, Y., Wang, Z. and Du, T.

Notes: In this study, in vivo selection of a phage display random peptide library was performed in mice, isolating specific peptides that homed to mouse thymus. A thymus-homing and kidney-recovered peptide were injected into BALB/c mice, and peripheral blood removed at intervals of 15 minutes, 1 hour, 3 hours, 6 hours and 24 hours. DNA was extracted using the ReadyAmp™ Genomic DNA Purification System. Thymus activity was assessed by using real-time PCR. (4020)

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