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Pcmv egfp

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PCMV-EGFP is a plasmid vector that contains the enhanced green fluorescent protein (EGFP) gene under the control of the cytomegalovirus (CMV) promoter. This plasmid can be used to express EGFP in mammalian cells.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Pcmv yap1 2α s127a, by Addgene (2 mentions) Pcmv yap1 2α s94a, by Addgene (2 mentions) Dulbecco modified eagle medium (dmem), by Corning (2 mentions) Hek293t cell, by American Type Culture Collection (2 mentions) Dual glo luciferase assay system, by Promega (2 mentions) Genehogs, by Thermo Fisher Scientific (2 mentions) Verteporfin, by Merck Group (1 mentions) Pgl3 basic luciferase reporter plasmid, by Promega (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Luciferase assay system, by Promega (1 mentions) Antibiotic antimycotic aa, by Thermo Fisher Scientific (1 mentions) Pci neo cova, by Addgene (1 mentions) Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions) Pureyield plasmid midiprep system, by Promega (1 mentions)

5 protocols using pcmv egfp

1

Yap isoform functional analysis via luciferase reporter

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Hek293T cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM; Cellgro) supplemented with 10% FBS (Gibco). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Hek293T cells were co-transfected with glutamine synthetase reporter constructs29 (link), the Renilla plasmid, and mutant isoforms of yap64 (link), 65 (link), including pCMV-Yap1-2α-S127A (Addgene, #17794), or pCMV-Yap1-2α-S94A (Addgene, #33102). pCMV-EGFP (Addgene, #11153) was used as control. Cells were harvested 48 hrs after transfection using the Dual-Glo Luciferase Assay System (Promega) and assayed according to the manufacturer's directions.
Cox A.G., Hwang K.L., Brown K.K., Evason K., Beltz S., Tsomides A., O'Connor K., Galli G.G., Yimlamai D., Chhangawala S., Yuan M., Lien E.C., Wucherpfennig J., Nissim S., Minami A., Cohen D.E., Camargo F.D., Asara J.M., Houvras Y., Stainier D.Y, & Goessling W. (2016). Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nature cell biology, 18(8), 886-896.
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2

Regulation of Ccl2 Promoter Activity

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HEK293 cells (ATCC CRL-3216) were transfected using Lipofectamine2000 reagent (Invitrogen) according to the manufacturer’s instructions. The 1.5 kb sequence immediately proximal to the first exon of the rat Ccl2 gene was cloned into the XhoI and HindIII sites of the pGL3 Basic luciferase reporter plasmid (Promega). Cells were co-transfected with pCMV-eGFP (Addgene #13031) or pCMV-YAP(S127A) (Addgene #27370), and treated with verteporfin (Sigma #SML0534) or DMSO vehicle for 24 h. Following transfection and treatments, cells were lysed with Passive Lysis Buffer (Promega), and luciferase activity was measured using the luciferase assay system (Promega) with an OPTOCOMP I luminometer (MGM Instruments). The luminescence was normalized by total protein content.
Francisco J., Zhang Y., Nakada Y., Jeong J.I., Huang C.Y., Ivessa A., Oka S., Babu G.J, & Del Re D.P. (2021). AAV-mediated YAP expression in cardiac fibroblasts promotes inflammation and increases fibrosis. Scientific Reports, 11, 10553.
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3

Yap isoform functional analysis via luciferase reporter

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Hek293T cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM; Cellgro) supplemented with 10% FBS (Gibco). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Hek293T cells were co-transfected with glutamine synthetase reporter constructs29 (link), the Renilla plasmid, and mutant isoforms of yap64 (link), 65 (link), including pCMV-Yap1-2α-S127A (Addgene, #17794), or pCMV-Yap1-2α-S94A (Addgene, #33102). pCMV-EGFP (Addgene, #11153) was used as control. Cells were harvested 48 hrs after transfection using the Dual-Glo Luciferase Assay System (Promega) and assayed according to the manufacturer's directions.
Cox A.G., Hwang K.L., Brown K.K., Evason K., Beltz S., Tsomides A., O'Connor K., Galli G.G., Yimlamai D., Chhangawala S., Yuan M., Lien E.C., Wucherpfennig J., Nissim S., Minami A., Cohen D.E., Camargo F.D., Asara J.M., Houvras Y., Stainier D.Y, & Goessling W. (2016). Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nature cell biology, 18(8), 886-896.
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4

Plasmid-Mediated Cell Transfection Protocols

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A RAW264.7 (murine macrophage) cell line was provided by Dr. Terry Connell (Department of Microbiology and Immunology, University at Buffalo, SUNY). The cell line was maintained in medium prepared as follows: 50 mL of FBS (heat inactivated), 5 mL of 100 mM MEM sodium pyruvate, 5 mL of 1 M HEPES buffer, 5 mL of penicillin/streptomycin solution, and 1.25 g of g D-(+)glucose added to 500 mL RPMI-1640 and filter sterilized. The HeLa (human epithelial) cell line was provided by Dr. Stelios Andreadis (Department of Chemical and Biological Engineering, University at Buffalo, SUNY) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) FBS (GIBCO) and 1% (v/v) Antibiotic-Antimycotic (A/A; Gibco). All cell lines were housed in T75 flasks and cultured at 37°C/5% CO2.
To determine in vitro transfection efficacy, we employed two reporter genes encoding luciferase and EGFP with each driven by a cytomegalovirus promoter within plasmids pCMV-Luc (Elim Biopharmaceuticals) and pCMV-EGFP (Addgene plasmid 11153[34 (link)]). Alternatively, in vivo transfection was evaluated using pCI-neo-cOVA (Addgene plasmid 25097[35 (link)]). Plasmids were transformed into and isolated from an E. coli cloning host (GeneHogs, Invitrogen) using a PureYield™ Plasmid Midiprep System (Promega) prior to being used in the experiments outlined below.
Jones C.H., Chen M., Ravikrishnan A., Reddinger R., Zhang G., Hakansson A.P, & Pfeifer B.A. (2014). Mannosylated poly(beta-amino esters) for targeted antigen presenting cell immune modulation. Biomaterials, 37, 333-344.
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5

Polymer-DNA Nanocomplex Formation

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Nanocomplexes were formed based on spontaneous electrostatic interactions between PEG-b-CPCL-70 and pCMV-EGFP pDNA at various polymer:pDNA mass ratios. The pDNAs were produced by an Escherichia coli bacterial host (GeneHogs, Invitrogen), with an enhanced green fluorescent protein reporter gene, driven by a cytomegalovirus promoter within plasmids (pCMV-EGFP, Addgene). A stock aqueous solution of polymer was prepared at a concentration of 4 mg/mL in ultrapure water. For each polymer:pDNA nanocomplex mass ratio, a specific amount of polymer solution was mixed with a pDNA solution, gently vortexed, and incubated for 30 min to ensure the formation of polymer:pDNA nanocomplexes. For example, for the preparation of nanocomplex at 200:1 of PEG-b-CPCL-70:pDNA mass ratio, 400 μL of polymer stock solution (4 mg/mL) was mixed with pCMV-EGFP pDNA (with a final concentration of 8 µg/mL) to a final volume of 1 mL.
Jafari A., Rajabian N., Zhang G., Alaa Mohamed M., Lei P., Andreadis S.T., Pfeifer B.A, & Cheng C. (2020). PEGylated Amine-Functionalized Poly(ε-caprolactone) for the Delivery of Plasmid DNA. Materials, 13(4), 898.
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