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Pef6 vector

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The PEF6 vector is a plasmid-based expression vector designed for gene expression in mammalian cells. It contains a strong viral promoter, a multiple cloning site for inserting target genes, and a selectable marker for antibiotic resistance.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Quikchange 2 site directed mutagenesis, by Agilent Technologies (1 mentions) Anti slp 76, by Cell Signaling Technology (1 mentions) Anti lat, by Merck Group (1 mentions) Anti zap70, by Cell Signaling Technology (1 mentions) 4d nucleofector system, by Lonza (1 mentions) Bxpc 3, by American Type Culture Collection (1 mentions) Penicillin, by Fujifilm (1 mentions) Streptomycin, by Fujifilm (1 mentions) Rpmi 1640, by Fujifilm (1 mentions) Amphotericin b suspension, by Fujifilm (1 mentions) Cdna synthesis kit, by Toyobo (1 mentions) Riboex, by GeneAll (1 mentions) Human recombinant insulin, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by American Type Culture Collection (1 mentions) Mycoalert mycoplasma detection kit, by Lonza (1 mentions) Transit brca transfection reagent, by Mirus Bio (1 mentions) L glutamine, by GenDEPOT (1 mentions) Plko 1 lentiviral vector, by Addgene (1 mentions)

Spelling variants of this product

PEF6/V5-His vector (8 citations) PEF6/V5-His TOPO vector (6 citations) PEF6/V5 His TOPO TA expression vector (4 citations) PEF6/Myc-His vector (4 citations) PEF6 expression vector (2 citations) PEF6/V5-His-TOPO™ expression vector (2 citations) PEF6/V5-His TOPO TA expression plasmid vector (2 citations) PEF6/V5-His-TOPO plasmid vector (2 citations) PEF6 mammalian expression vector (2 citations) PEF6/His TOPO vector (2 citations)

8 protocols using pef6 vector

1

Csk Variant Construction and Tagging

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Full-length cDNA encoding mouse Csk was subcloned into pEF6 vector (Life Technologies) with a C-terminal linker SAGGSAGG66 (link) followed by a Myc (EQKLISEEDL) or HA (YPYDVPDYA) epitope tag. The variants H21I, K43D, W47A46 (link), and K222R3 (link) were created using QuikChange II site-directed mutagenesis (Agilent).
Brian BF I.V., Sjaastad F.V, & Freedman T.S. (2022). SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding. Scientific Reports, 12, 5875.
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2

Jurkat T-cell Line Variants and Receptor Expression

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The Jurkat E6.1 T-cell line and its Zap70- (P116) and Lat-(JCaM2.5) deficient variants were kindly provided by A. Weiss (UCSF, San Francisco, USA). A chimeric cDNA construct coding for the extracellular and transmembrane regions of human CD25 linked to the cytoplasmic region of mouse CD3ξ and termed CD25ξ has been described50 (link). A full-length cDNA corresponding to the largest isoform of human CD6 (CD6-201 Ensembl ENST00000344028) was isolated from Jurkat cells by reverse transcription and cloned into the pEF6 vector (Life Technology). Stable transfectants expressing CD25ξ chimeric constructs and human CD6 molecules were obtained by electroporation of Jurkat cells and sorted based on their level of CD6 or CD25ξ expression. Stimulating antibodies against CD25 (7G7) were from Millipore. Anti-ZAP70 (2705; Cell Signaling Technology), anti-SLP76 (4958; Cell Signaling Technology), anti-LAT (06-807; Millipore), anti-GADS (06-983; Millipore), anti-CD6 (H300; Santa Cruz Biotechnology), and antibodies to phosphorylated tyrosines (4G10; Millipore) were used for immunoblot analysis.
Roncagalli R., Hauri S., Fiore F., Liang Y., Chen Z., Sansoni A., Kanduri K., Joly R., Malzac A., Lähdesmäki H., Lahesmaa R., Yamasaki S., Saito T., Malissen M., Aebersold R., Gstaiger M, & Malissen B. (2014). Quantitative proteomic analysis of signalosome dynamics in primary T cells identifies the CD6 surface receptor as a Lat-independent TCR signaling hub. Nature immunology, 15(4), 384-392.
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3

Cloning and Expression of HHLA2-mIgG2a Fusion Protein

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The coding region of human HHLA2 was PCR-amplified using the primers 5’-TGTTCTGCACAAGACA-3’ (sense primer located just 5’ of ATG start) and 5’-GTAAGGATGCAGGTCATGAGT-3’ (anti-sense primer located just 3’ to stop codon) and introduced into the pEF6 vector (Thermo Fisher; cat. #K961020) by TA cloning.
The HHLA2-mIgG2a fusion protein was made by fusing cDNA’s for the HHLA2 extracellular domain to the mouse IgG2a hinge and Fc domains (with mutations to reduce binding to Fc receptors) and cloning into the pEF6 vector. The construct was introduced into CHO cells and HHLA2-mIgG2a fusion protein was purified by affinity chromatography on a protein G column.
Bhatt R.S., Berjis A., Konge J.C., Mahoney K.M., Klee A.N., Freeman S.S., Chen C.H., Jegede O.A., Catalano P.J., Pignon J.C., Sticco-Ivins M., Zhu B., Hua P., Soden J., Zhu J., McDermott D.F., Arulanandam A.R., Signoretti S, & Freeman G.J. (2020). KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1. Cancer immunology research, 9(2), 156-169.
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4

Stable Cell Lines Expressing Inhibitory Biologics

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To generate the plasmids, gBlocks gene fragments (IDT) encoding the inhibitory biologic (FLAG-mCherry2-GGSGGS-SIWWPD) and the control biologic (FLAG-mCherry2-GGSGGS-SIWWHR) were cloned into pEF6 vector (Thermo Fisher Scientific) by standard restriction enzyme digestion and T4 DNA ligation. The final constructs were verified by Sanger sequencing. Coding sequences of the constructs are provided in Supplementary Table S5. To generate the stable cell lines, a total of 2 × 105 B16-F10, A375 or A549 cells were electroporated with 200 ng plasmid using DJ-110, FF-120 or CM-130 respectively—the preset programmed in 4D-nucleofector system (Lonza). The preparation of the cell suspension and the process of the electroporation are similar to that described in the CRISPR method section. The stable cell lines were selected using 10 µg ml−1 blasticidin three days post-electroporation and were maintained in 5 µg ml−1 blasticidin once the stable colonies were established.
Ng S., Lim S., Sim A.C., Mangadu R., Lau A., Zhang C., Martinez S.B., Chandramohan A., Lim U.M., Ho S.S., Chang S.C., Gopal P., Hong L.Z., Schwaid A., Fernandis A.Z., Loboda A., Li C., Phan U., Henry B, & Partridge A.W. (2022). STUB1 is an intracellular checkpoint for interferon gamma sensing. Scientific Reports, 12, 14087.
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5

Generating Murine Pancreatic Cancer Cells

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LTPA, a murine pancreatic cancer cell line, and BxPC3, a human pancreatic cell line, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). To obtain LTPA cells showing high expression levels of mouse TF (mTF) (LTPA-TF), the mTF gene was cloned into a pEF6 vector (Invitrogen, Carlsbad, CA, USA), followed by transfection of the LTPA cells with the expression vector. LTPA-TF cells stably expressing mTF were obtained by selection using the drug blastcidin. The cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 10% FBS (Gibco, NY, USA) and 100 units/mL Penicillin, 100 μg/mL Streptomycin and 0.25 μg/mL Amphotericin B suspension (Wako) in an atmosphere containing 5% CO2 at 37°C.
Sato R., Obonai T., Tsumura R., Tsumoto K., Koga Y., Yasunaga M, & Matsumura Y. (2014). Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis. Cancer Science, 105(12), 1631-1637.
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6

Overexpression of TEN1 and HCV NS5A

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Total cellular RNAs were extracted from Huh7.5 cells by using RiboEx (GeneAll Biotechnology, Korea), and cDNA was synthesized by using a cDNA synthesis kit (Toyobo, Japan) according to the manufacturer’s instructions. Full-length TEN1 was amplified by a primer set (Table 1). Polymerase chain reaction (PCR) products were inserted into the HindIII and EcoRI sites of the p3XFLAG-CMV10 vector (Invitrogen, USA). siRNA-resistant TEN1 mutant was constructed by introducing two silent mutations in the siRNA-binding site. cDNA corresponding to the NS5A coding sequence of HCV was amplified by PCR using the Korean isolate of HCV (genotype 1b) and subcloned into the pEF6 vector (Invitrogen).
Lim Y.S., Nguyen M.T., Pham T.X., Huynh T.T., Park E.M., Choi D.H., Kang S.M., Tark D, & Hwang S.B. (2021). Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV. Molecules and Cells, 45(3), 148-157.
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7

Genetic Manipulation of T47D Cells

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T47D cells (ATCC HTB-13) were maintained in RPMI-1640 medium (30–2001, ATCC) supplemented with 10 μg/mL human recombinant insulin (12585–014, Gibco), 2 mM L-glutamine (CA009–010, GenDepot) and 10% FBS (16000044, Life Technologies). Cells were transfected with mGFP-KRASG12V, -HRASG12V or -LactC2 in pEF6 vector (Invitrogen) using TransIT-BrCa Transfection Reagent (MIR 5500, Mirus), and selected with 10 μg/mL blasticidin for 3 weeks. After the selection, cells were maintained in 5 μg/mL blasticidin. For generating MTMR knockdown cell lines, wild-type (WT) T47D cells were infected with lentivirus expressing shRNA targeting MTMR2, 3, 4 or 7. Cells were selected by puromycin (1 μg/mL) for 1 week and maintained in 0.5 μg/mL puromycin. Cells were grown at 37°C in 5% CO2 and frequently tested for mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza, LT07–318).
Henkels K.M., Miller T.E., Naji A., van der Hoeven R., Liang H., Zhou Y., Hammond G.R., Hancock J.F, & Cho K.J. (2024). Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine. bioRxiv.
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8

Smad7 Overexpression and Knockdown

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pEF1α-BirA-V5 construct expresses C-terminal V5-tagged BirA biotin ligase. The full-length Smad7 cDNA and mutant (Smad7Δ407; it lacks 19 amino acids at its C-terminus) were obtained by PCR amplification and N-terminally tagged by Avitag. Then the expression constructs were cloned into the Not I and Xba I sites of the pEF6 vector (Invitrogen, Waltham, MA, USA).
Smad7 shRNAs were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search (accessed on 17 December 2021)) targeting the following sequences:
5′-GCTTTCAGATTCCCAACTTCT-3′ (shRNA1 Smad7);
5′-GTCTTGTTCTTTGAGAAATTA-3′ (shRNA2 Smad7)
Annealed oligonucleotides were cloned into pLKO.1 lentiviral vector (Addgene:10879), and each construct was verified by sequencing.
Meng G., Lauria A., Maldotti M., Anselmi F., Polignano I.L., Rapelli S., Donna D, & Oliviero S. (2021). Genome-Wide Analysis of Smad7-Mediated Transcription in Mouse Embryonic Stem Cells. International Journal of Molecular Sciences, 22(24), 13598.
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