Pcdh ef1 mcs t2a copgfp

Manufactured by System Biosciences

Sourced in United States

The PCDH-EF1-MCS-T2A-copGFP is a plasmid vector that contains a multi-cloning site (MCS) downstream of the PCDH promoter and the EF1 enhancer. The T2A self-cleaving peptide sequence is used to link the gene of interest to a copGFP reporter, allowing for visualization of transgene expression.

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Lab products found in correlation

T7 and cmv promoters, by Promega (1 mentions) E coli krx, by Promega (1 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions) Mcherry, by Genewiz (1 mentions) Polyethylenimine (pei), by Polysciences (1 mentions) Pcmvr8, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions)

8 protocols using pcdh ef1 mcs t2a copgfp

Site-Directed Mutagenesis and Protein Expression

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CBR mutants/mutant libraries were constructed using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies 200523) according to the manufacturer protocol. Oligonucleotides were from IDT. Mutagenesis reactions were used to transform E. coli KRX (Promega). Individual colonies were picked for plasmid preparation and DNA sequence verification. All plasmids for bacterial expression and transient mammalian cell expression were in a pF4Ag backbone (T7 and CMV promoters; Promega). For purification from bacterial overexpression, sequences were sub-cloned to pF6HisNK (Promega). CBR2opt was assembled synthetically as a mammalian codon optimized version of CBR2 expressing the identical enzyme sequence as CBR2 (Gene Dynamics). For stable cell line generation, Luc2 and CBR2opt were sub-cloned from their pF4Ag backbones into lentiviral vector pCDH-EF1-MCS-T2A-copGFP (System Biosciences).

Hall M.P., Woodroofe C.C., Wood M.G., Que I., van’t Root M., Ridwan Y., Shi C., Kirkland T.A., Encell L.P., Wood K.V., Löwik C, & Mezzanotte L. (2018). Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging. Nature Communications, 9, 132.

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Generation of G-THP-1 Cells via Lentiviral Transduction

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A THP-1 cell line that expresses GFP (G-THP-1) was generated though lentiviral transduction. Vectors that contain plasmids of pSL3 (vesicular stomatitis virus G envelope), pSL4 (HIV-1 gag/pol packing genes), pSL5 (rev gene required for HIV-1 envelope protein expression) were a gift from Dr. Murry (University of Washington, Seattle, WA). The plasmid vector pCDH-EF1-MCS-T2A-copGFP was purchased from System Biosciences. The lentiviral vector was packaged in HEK293T cells as previously described. Briefly, 3×106 of HEK293T cells were seeded in 10-cm dishes to reach 60% confluence in DMEM with 10% FBS and 100U/mL penicillin-streptomycin. The culture media was changed prior to transfection. A total of 20ug plasmid DNA (7.5 ug pCDH-EF1-MCS-T2A-copGFP, 2.5 ug pSL3, 6.7 ug pSL4, 3.3 ug pSL5) and 24 uL of lipofectamine 2000 dissolved in 1600 uL Opti-MEM were used for the transfection of one dish. The media was replaced after incubating for 6 hours at 37°C with 5% CO2. The virus supernatant was collected at 48 hours from the media and filtered through a 0.45-μm filter. The media containing the virus was applied to the THP-1 cells at a multiplicity of infection of 6 while spinning at 800×g for 30 minutes. The transduced THP-1 cells (G-THP-1) were allowed to expand and sorted for GFP expression using a to obtain over 92% transduction efficiency.

Zhou H., Tu L.N., Giachelli C., Nigam V, & Scatena M. (2023). Monocyte Adhesion and Transmigration Through Endothelium Following Cardiopulmonary Bypass Shearing is Mediated by IL-8 Signaling. bioRxiv.

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Visualizing KIR Endosomal Localization

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To disrupt human μ2 (NM_001025205; ATCC, Manassas, VA) binding to tyrosine-based motifs, residues D176 and W420 were mutated to alanine [μ2-dominant negative (DN)]. To visualize KIR localization within the endosomal compartment, 3DL1*001 fused in-frame to mCherry (Genewiz, South Plainfield, NJ) and EYFP-Rab4 or EGFP-Rab7 (generous gift from Dr. Mario Zerial, Max Planck Institute, Dresden, Germany) were subcloned into pBMN-NoGFP to generate retrovirus as described (26 , 34 ). For primary NK cell expression, constructs were subcloned into pCDH-EF1-MCS-T2A-copGFP (System Biosciences, Mountain View, CA) to generate lentivirus by transfecting LentiX 293T cells with pCDH, pMD2.G (VSV-G) and psPAX2 (gag/pol) plasmids (from Dr. Sam Kung, University of Manitoba, Winnipeg). Lentiviral supernatants were harvested 48–72 hr later, filtered and concentrated by ultracentrifugation or PEG precipitation. Viral titers were determined in LentiX 293T cells (35 (link)). Primary NK cells were infected on two consecutive days with lentivirus (MOI of 20–40 in 8 μg/ml polybrene). To alleviate compensatory mechanisms resulting from long-term expression of exogenous proteins, cells were assayed 48–72 hr after the second infection.

Purdy A.K., Alvarez-Arias D.A., Oshinsky J., James A.M., Serebriiskii I, & Campbell K.S. (2014). THE AP-2 CLATHRIN ADAPTOR MEDIATES ENDOCYTOSIS OF AN INHIBITORY KILLER CELL Ig-LIKE RECEPTOR (KIR) IN HUMAN NK CELLS. Journal of immunology (Baltimore, Md. : 1950), 193(9), 4675-4683.

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Cloning CLIP2 into Lentiviral Vector

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CLIP2 was amplified from TPC-1 cDNA using gene specific primers (Forward: 5′-TCTAGAGAATTCGCCACCATGCAGAAGCCCAGCGGCCTGAAG-3′; Reverse: 5′.
TCTAGATAGCGGCCGCGTGCTTGTCCTCTTGTTTCTGAGC-3′) and cloned into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP (System Biosciences, Mountain View, CA, USA). The sequence of the inserted CLIP2 fragment was confirmed by sequencing (NCBI reference sequence: NM_032421.2).

Selmansberger M., Michna A., Braselmann H., Höfig I., Schorpp K., Weber P., Anastasov N., Zitzelsberger H., Hess J, & Unger K. (2020). Transcriptome network of the papillary thyroid carcinoma radiation marker CLIP2. Radiation Oncology (London, England), 15, 182.

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Lentiviral Transduction of TCL1 Leukemic Cells

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The sequence encoding ovalbumin (Addgene, cat. number 25097) was inserted into mammalian expression vector pCDH-EF1-MCS-T2A-copGFP (System Biosciences). The pCDH-EF1-OVA-GFP and a packaging (psPAX2) and an envelope (pMD2.G) plasmids (gifts from prof. Didier Trono, École Polytechnique Fédérale de Lausanne, Switzerland) were introduced into HEK-293T cells using Polyethylenimine (Polysciences). Then freshly isolated TCL1 cells (CD5⁺CD19⁺) from mouse spleens were seeded into 24-well plates with M2-10B4 murine stroma cells. Next, medium containing lentiviral particles was added into TCL1 and M2-10B4 cells co-culture. Then TCL1 cells were washed and inoculated into RAG2-KO mice for leukemic cells propagation. Finally, OVA⁺ GFP⁺ cells were sorted and used for further experiments. In all performed experiments at least 60% of injected leukemic cells exerted OVA⁺ GFP⁺ phenotype as evaluated by flow cytometry.

Goral A., Firczuk M., Fidyt K., Sledz M., Simoncello F., Siudakowska K., Pagano G., Moussay E., Paggetti J., Nowakowska P., Gobessi S., Barankiewicz J., Salomon-Perzynski A., Benvenuti F., Efremov D.G., Juszczynski P., Lech-Maranda E, & Muchowicz A. (2022). A Specific CD44lo CD25lo Subpopulation of Regulatory T Cells Inhibits Anti-Leukemic Immune Response and Promotes the Progression in a Mouse Model of Chronic Lymphocytic Leukemia. Frontiers in Immunology, 13, 781364.

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Lentiviral Expression of Syt1 Variants

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The full-length rat Syt1^WT, Syt1^6DA or Syt1^D238N mutant was amplified by PCR using primers: 5’- ATGCGGGCCCATGGTGAGTGCCAGTCATC-3’ and 5’- TGTTGTCGACTTACTTCTTGACAGCCAGCATG-3’, and introduced into a lentiviral expression vector pCDH-EF1-MCS-T2A-copGFP (System Biosciences, catalogue number: CD526A-1) between the ApaI and the SalI sites. Lentiviral infections with Syt1^WT, Syt1^6DA or Syt1^D238N in Syt1 KO cells were carried out on chromaffin cells at days in vitro 0, and electrophysiological recordings were carried out 2–3 days after infection as described (Jiang et al. 2019 (link)). All the custom-made materials will be shared upon reasonable requests.

Varga K., Jiang Z, & Gong L.W. (2019). Phosphatidylserine is critical for vesicle fission during clathrin-mediated endocytosis. Journal of neurochemistry, 152(1), 48-60.

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Constructing Lentiviral SphK1 Overexpression Vectors

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The protein-coding cDNA clone of mouse SphK1, clone ID pCS6 (BC037710), was produced with transOMIC technologies. To obtain pCDH-EF1-SphK1-T2A-copGFP, the full-length SphK1 was amplified by PCR using primers: 5’-ACTCTCTAGAATGGAACC AGTAGAATGCC-3’ and 5’- GATTGCGGCCGCTAATGGTTCTTCTGGAGGTG-3’, and introduced into a lentiviral expression vector pCDH-EF1-MCS-T2A-copGFP (System Biosciences, catalogue number: CD526A-1) between the XbaI and the NotI sites. For Ca²⁺ imaging experiments presented in Fig. S1, the full length of SphK1 was introduced into the lentiviral expression vector with a N-terminal FLAG tag we created (pCDH-EF1-N-FLAG-MCS) between the XbaI and the NotI sites. The SphK-1^DN mutation (Pitson et al. 2000 (link), Bonhoure et al. 2006 (link), Gomez-Brouchet et al. 2007 (link)) was made by QuikChange PCR (Stratagene) targeting the mutation G82D using primers: 5’-GTCATGTCCGGTGATGATCTGATGCATGAGGTG-3’ and 5’-CACCTCATGCATCAGATC ATCACCGGACATGAC-3’. The full length SphK1 and G82D mutation were verified by Sanger sequencing.

Jiang Z.J., Delaney T.L., Zanin M.P., Haberberger R.V., Pitson S.M., Huang J., Alford S., Cologna S., Keating D.J, & Gong L.W. (2019). Extracellular and intracellular sphingosine-1-phosphate distinctly regulates exocytosis in chromaffin cells. Journal of neurochemistry, 149(6), 729-746.

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Lentiviral Transduction of Immune Cells

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Human embryonic kidney–293T cells were transfected with the respective constructs cloned into pCDH-EF1-MCS-T2A-copGFP (System Biosciences) and the packaging plasmids pMD2.G and pCMVR8.74 (Addgene) using the transfection reagent polyethylenimine. The virus-containing supernatant was used for transduction of the immune cell lines.

Marasco M., Berteotti A., Weyershaeuser J., Thorausch N., Sikorska J., Krausze J., Brandt H.J., Kirkpatrick J., Rios P., Schamel W.W., Köhn M, & Carlomagno T. (2020). Molecular mechanism of SHP2 activation by PD-1 stimulation. Science Advances, 6(5), eaay4458.

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