Pdisplay vector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PDisplay vector is a plasmid vector designed for protein expression in mammalian cell lines. It provides a strong promoter and a polylinker for inserting the gene of interest. The vector contains elements for selection and maintenance in both bacterial and mammalian cells.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Cobalt agarose resin, by Thermo Fisher Scientific (2 mentions) Lsm 700, by Zeiss (2 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (2 mentions) T5 exonuclease, by New England Biolabs (2 mentions) Axioscop 2 plus, by Zeiss (2 mentions) Mouse monoclonal 12ca5 anti ha antibody, by Abcam (2 mentions) Fitc goat anti mouse ig, by BD (2 mentions) Anti c myc 9e10 monoclonal antibodies, by Abcam (2 mentions) Goat anti mouse igg texas red, by Thermo Fisher Scientific (2 mentions) Facscanto 2 cytometer, by BD (2 mentions) Pdisplay expression construct, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions) Metamorph software, by Molecular Devices (1 mentions) Itaq universal sybr green kit, by Bio-Rad (1 mentions) Rotor gene 3000 cycler, by Qiagen (1 mentions) Hek293 cells, by Promega (1 mentions) Hek293 cells, by Japanese Collection of Research Bioresources Cell Bank (1 mentions) Ez link sulfo nhs lc biotin kit, by Thermo Fisher Scientific (1 mentions) Quikchange lightning mutagenesis kit, by Agilent Technologies (1 mentions)

Spelling variants of this product

PDisplay (14 citations)

35 protocols using pdisplay vector

Generating Lentiviral Constructs with YFP/GLuc Fusion Protein

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Lentiviral constructs were generated using standard molecular biology techniques (Figure 1). The YFP/GLuc fusion protein was cloned into a lentiviral backbone (LV.YFP/GLuc). The pDisplay™ vector (Life Technologies, Invitrogen, Carlsbad, CA, USA), a vector that anchors any protein to the cell membrane, was cloned into this YFP/GLuc lentiviral backbone, yielding the LV.MT-YFP/GLuc vector. A control vector was prepared by cloning the GLuc gene into a lentiviral plasmid containing a red fluorescent fluorochrome (Red/GLuc).

Goethals L.R., Bos T.J., Baeyens L., De Geeter F., Devoogdt N, & Lahoutte T. (2014). Camelid reporter gene imaging: a generic method for in vivo cell tracking. EJNMMI Research, 4, 32.

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Purification of His-tagged IL-15/IL-15Rα

Cited 1 time

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We generated a pDisplay Vector (Life Technologies) encoding His-tagged IL-15/IL-15Rα (IL-15 linked to IL-15P sushi)^{28 (link)}. Following transfection of 293-F cells, we purified protein from culture supernatant using standard Cobalt agarose resin (Thermo Scientific) according to protocol.

Stephan S.B., Taber A.M., Jileaeva I., Pegues E.P., Sentman C.L, & Stephan M.T. (2014). Biopolymer implants enhance the efficacy of adoptive T cell therapy. Nature biotechnology, 33(1), 97-101.

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Purification of His-tagged IL-15/IL-15Rα

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Stephan S.B., Taber A.M., Jileaeva I., Pegues E.P., Sentman C.L, & Stephan M.T. (2014). Biopolymer implants enhance the efficacy of adoptive T cell therapy. Nature biotechnology, 33(1), 97-101.

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Constructing Mammalian Expression Vectors

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To construct mammalian expression vectors, the full-length genes of the ZnGreen1, ZnGreen2, and ZnRed sensors were amplified from their corresponding E. coli expression vectors with oligo pairs pCMV-ZnGreen1-F and pCMV-ZnGreen1-R, pCMV-ZnGreen2-F and pCMV-ZnGreen2-R, and pCMV-ZnRed-F and pCMV-ZnRed-R, respectively. The amplified PCR products were digested with Hind III and Xho I and ligated into a pre-digested pcDNA3-pnGFP-WPRE plasmid^{30 (link)}. To construct vectors with nuclear localization, oligos pNuc-ZnRed-F and pNuc-ZnRed-R were used to amplify the ZnRed gene from plasmid pTorPE-ZnRed. PCR products were then digested with Nhe I and Xho I, and ligated into a predigested phsGFP-Nuc plasmid ^{31 (link)} to create pNuc-ZnRed, which contains three copies of the nuclear localization signal (NLS) sequence (DPKKKRKV) at its C-terminus. To localize ZnGreen1 sensor to the extracellular cell surface, a modified pDisplay vector (Life Technologies, Carlsbad, CA) was first amplified with primers pDisplay-Vector-F and pDisplay-Vector-R. The ZnGreen1 gene was then amplified with primers ZnGreen1-Display-F and ZnGreen1-Display-R, and inserted into the pDisplay vector by the Gibson Assembly Cloning method^{32 (link)} to create pDisplay-ZnGreen1. Plasmid identities were verified by DNA sequencing. Sensor localizations were confirmed by fluorescence microscopy of transfected cells.

Chen Z, & Ai H.W. (2016). Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn2+). Analytical chemistry, 88(18), 9029-9036.

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Cloning and Expression of PPRV ICV Protein

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The sequence of the head domain of PPRV ICV’89 strain H protein (residues 156–610) was amplified by PCR using specific primers from a previously reported construction obtained in our lab (pSIREN-H) as template [22 (link)]. The fragment was BamHI/XhoI digested and ligated into a pDisplay vector (Life Technologies). A C-terminal stop codon was introduced to avoid protein fusion to the PDGFR transmembrane domain. The resulting construction (pDisplay-H-ICV) contains an N-terminal fusion of the protein with the murine Igκ-chain leader sequence, which directs it to the secretory pathway, followed by an HA tag to allow purification. The construction was proof-sequenced to confirm its identity.

Rojas J.M., Pascual E., Wattegedera S.R., Avia M., Santiago C., Martín V., Entrican G, & Sevilla N. (2021). Hemagglutinin protein of Peste des Petits Ruminants virus (PPRV) activates the innate immune response via Toll-like receptor 2 signaling. Virulence, 12(1), 690-703.

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Molecular Engineering of Lamprey BAFF-like and BCMAL Receptors

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293T cells were cultured in DMEM (sigma) containing 5% FBS at 37° C and 5% CO₂. Lamprey BAFF-like (BAFFL) ligand and human ectodysplasin A was ligated into the pDisplay vector (Thermo Fisher Scientific), fused with N-terminal hemagglutinin (HA) tag (pDisplay-HA-BAFFL and pDisplay-HA-hEDA). Lamprey BCMAL1 and BCMAL2 receptors are also ligated into the pDisplay vector, fused with N-terminal FLAG-tag (pDisplay-FLAG-BCMAL1 and pDisplay-FLAG-BCMAL2). The extracellular domain of lamprey BCMAL-receptors were also fused with human IgG Fc-tag at the C-terminal, and ligated in pIRES puro vector (pIRES puro-FLAG-BCMAL1-Fc and pIRES puro-FLAG-BCMAL2-Fc). The expression vectors were transfected to 293T cells using polyethylenimine (PEI). After three days incubation, the culture medium and transfected cells were harvested for further assay.

Das S., Sutoh Y., Cancro M.P., Rast J.P., Han Q., Bommakanti G., Cooper M.D, & Hirano M. (2019). Ancient BCMA-like genes herald B-cell regulation in lampreys. Journal of immunology (Baltimore, Md. : 1950), 203(11), 2909-2916.

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Analyzing SALM3 Mutant Effects on Synaptic Markers

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HEK293T cells were transfected with the indicated mutants and the WT SALM3 cloned in pDisplay vector (Thermo Fisher Scientific; listed in Table S4). After 48 h, transfected HEK293T cells were trypsinized, seeded onto cultured hippocampal neurons at DIV9, and then coimmunostained with antibodies against synapsin^{49 (link)} at DIV11. Images were acquired by confocal microscopy (LSM700, Carl Zeiss). For quantifications, the contours of transfected HEK293T cells were chosen as the ROI. The fluorescence intensities of synaptic marker puncta, normalized with respect to the area of each HEK293T cell were quantified for both red and green channels using MetaMorph Software (Molecular Devices). Surface expression was verified as explained in the Supplementary Fig. S10. All data are expressed as means ± SEM. All experiments were repeated using at least three independent cultures and data were evaluated statistically using a nonparametric Kruskal–Wallis test, followed by Dunn’s multiple-comparison test for post hoc groups comparisons (n values used are indicated in the figure legends). Significance was indicated with an asterisk (compared with a value from control group) or hashtag (compared with a value from SALM3 WT-group).

Karki S., Shkumatov A.V., Bae S., Kim H., Ko J, & Kajander T. (2020). Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTPσ. Scientific Reports, 10, 11557.

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Recombinant Expression of Influenza B HA1

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The polypeptide encoding HA1 region of B/Massachusetts/2/2012 virus was expressed on 293T cells as follows. The DNA segment encoding HA1 region was prepared by PCR using artificially constructed HA gene B/Massachusetts/2/2012 as template DNA and two primers SfiI FluBMas12HA F primer 5′-TTAGGCCCAGCCGGCCGACAGAATATG and FluBMas12HA345 XbaR primer 5′-GTCTCTAGACTCCTTCAGCAGTTTAGC. Then, HA1 with Flag tag was inserted into pDisplay vector (Thermo Fisher Scientific), resulting in BMas12HA1/pDisplay.

Hirano D., Ohshima N., Kubota-Koketsu R., Yamasaki A., Kurosawa G., Okuno Y., Yoshida S, & Kurosawa Y. (2018). Three Types of Broadly Reacting Antibodies against Influenza B Viruses Induced by Vaccination with Seasonal Influenza Viruses. Journal of Immunology Research, 2018, 7251793.

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Quantitative Analysis of ADAM10 Expression

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The hAdam10 D + C domains (AA454–673) were amplified by PCR and cloned into the pDisplay vector (Thermofisher, Waltham, MA, USA) using BglII and SalI restriction enzyme sites (retaining the N terminal HA tag and Myc at the membrane). The construct was transfected into HEK293 cells (Fugene, Promega), and a stable line was generated through antibiotic (neomycin) selection.
For quantitative PCR, cDNA generated from RNA extracts (QIAGEN RNeasy) of snap-frozen tumor samples was analysed using an iTaq Universal SYBR Green kit (Bio-Rad Laboratories, Hercules, CA, USA) and a RotorGene 3000 cycler (Corbett Research). Primers specific for human ADAM10 were used to determine expression by comparative CT (ΔΔCT), relative to the averaged expression of three housekeeping genes (GAPDH, Beta 2 microglobulin, and Eukaryotic translation initiation factor 4E).

Yan H., Vail M.E., Hii L., Guo N., McMurrick P.J., Oliva K., Wilkins S., Saha N., Nikolov D.B., Lee F.T., Scott A.M, & Janes P.W. (2022). Preferential Antibody and Drug Conjugate Targeting of the ADAM10 Metalloprotease in Tumours. Cancers, 14(13), 3171.

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Fcrl5 Monoclonal Antibody Generation

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The full-length coding sequence of Fcrl5 was cloned into pDisplay vector (Thermo Fisher Scientific) and then transfected into HEK-293 cells (JCRB Cell Bank) to establish a stable transfectant line (293-Fcrl5 cells). Wistar rats were immunized with the 293-Fcrl5 cells, and hybridomas were generated by fusing splenic B cells with mouse X-63 myeloma cells. The supernatants of each hybridoma were screened by ELISA using 293-Fcrl5 cells as antigens and further evaluated by flow cytometric analysis using a stable transfectant line expressing Fcrl5 in ST486 cells (ATCC). The antibody specificity was also evaluated using stable transfectants of Fcrl1 and Fcrl2 in HEK-293 cells, and the clone 128 was selected. The 128 hybridoma cells were injected into the peritoneal cavity of Pristan-primed BALB/c mice, and antibodies in the ascitic fluid were purified by protein A column chromatography. F(ab’)₂ fragment antibodies were generated by pepsin digestion of whole IgG antibodies. All the work from rat immunization to antibody fragmentation was conducted at Immuno-Biological Laboratories Co, Ltd. These antibodies were biotinylated using the EZ-Link Sulfo-NHS-LC-Biotin Kit (Thermo Fisher Scientific).

Ono C., Tanaka S., Myouzen K., Iwasaki T., Ueda M., Oda Y., Yamamoto K., Kochi Y, & Baba Y. (2023). Upregulated Fcrl5 disrupts B cell anergy causes autoimmune disease. Frontiers in Immunology, 14, 1276014.

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