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Pcag mgfp

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The PCAG-mGFP is a plasmid designed for the expression of mGFP (monomeric green fluorescent protein) in mammalian cells. It contains the CAG promoter, which drives high-level expression of the mGFP gene. The plasmid also includes a selectable marker for antibiotic resistance, allowing for the selection of transfected cells.

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Lab products found in correlation

Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Pef6v5 egfp caax 2a mcherry, by Addgene (1 mentions) Pcs membrane ceruleanfp, by Addgene (1 mentions) Pmirglo vector, by Promega (1 mentions) 3xflagnicd1, by Addgene (1 mentions) Fugene hd, by Promega (1 mentions) Dual glo luciferase assay system, by Promega (1 mentions)

Spelling variants of this product

PCAG-mGFP-actin (7 citations)

6 protocols using pcag mgfp

1

Lentiviral Vector Production Protocol

Cited 1 time
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The mGFP sequences from pCAG-mGFP (Addgene) were cloned and newly inserted under the EF1 promoter sequence [41 (link)]. Recombinant lentiviral vectors were produced by transient transfection of three plasmids, pCAG-HIVgp, pCMV-VSV-G-RSV-Rev, and the lentiviral vector plasmid (CSII-EF-mGFP), into HEK293T cells as previously described [42 (link), 43 (link)].
Culture supernatant containing lentivirus was concentrated by ultracentrifugation (50,000 G for 2 h at 4 ºC), and the viral pellets were resuspended in HBSS (14,025,092; Thermo Fisher Scientific). Harvested lentiviruses were stored at -80 ºC before use. The multiplicity of infection (MOI) values were adjusted to 15 based on the total cell numbers.
Nishijima T., Okuyama K., Shibata S., Kimura H., Shinozaki M., Ouchi T., Mabuchi Y., Ohno T., Nakayama J., Hayatsu M., Uchiyama K., Shindo T., Niiyama E., Toita S., Kawada J., Iwamoto T., Nakamura M., Okano H, & Nagoshi N. (2024). Novel artificial nerve transplantation of human iPSC-derived neurite bundles enhanced nerve regeneration after peripheral nerve injury. Inflammation and Regeneration, 44, 6.
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2

Generation of HDR Targeting Vectors

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To generate the HDR templates, the left and right homology arms (HAs) of each locus were amplified from the wild-type (WT) hiPSC genomic DNA (see Supplementary Table S2). The amplified HAs of VSX2, BRN3b, and RCVRN genes were inserted into the previously assembled vectors FRT.TK.Puro.FRT.pL451, LoxP.TK.Blast.LoxP.pL452, and FRT.TK.Neo.FRT.pL451 via Gibson Assembly (E2611L18 (link); NEB), respectively (vector sequences available upon request). These insertions were confirmed by DNA sequencing. The Cerulean fluorescent reporter gene was derived from pCS-membrane-CeruleanFP, a gift from Sean Megason (Addgene plasmid 53749).19 (link) The GAP43-eGFP reporter gene was derived from pCAG-mGFP, a gift from Connie Cepko (Addgene plasmid 14757).20 (link) The mCherry reporter gene was derived from pEF6V5:eGFP-CAAX-2A-mCherry, a gift from Steven Leach (Addgene plasmid 26901).21 (link)
Lam P.T., Gutierrez C., Del Rio-Tsonis K, & Robinson M.L. (2020). Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types. Translational Vision Science & Technology, 9(3), 21.
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3

Construct Viral Vectors for Neuronal Manipulation

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The AAV-ITR plasmid for DREADD pAAV-hSyn-DIO-hM4Di-mCherry was provided by Brian Roth (Krashes et al., 2011). The AAV-ITR plasmid for axonal tracing pAAV-EF1α-DIO-palGFP was constructed by subcloning the coding region of palGFP (mGFP) from pCAG-mGFP (a gift from Connie Cepko; Addgene plasmid #14757)108 (link) into the pAAV-EF1α-DIO cassette made from pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA (a gift from Karl Deisseroth; Addgene plasmid #20297).
Nakatsuka D., Kanda T., Sato M., Ishikawa Y., Cherasse Y, & Yanagisawa M. (2024). A novel GABAergic population in the medial vestibular nucleus maintains wakefulness and gates rapid eye movement sleep. iScience, 27(3), 109289.
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4

Plasmid Constructs for Gene Expression

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The pEF1α-EGFP plasmid expressing nuclear EGFP has been previously reported56 (link). Glo1 and control shRNAs in the pSUPER vector were published previously20 (link). The shRNAs against human GLO1 (GATGGCTACTGGATTGAAA) were cloned into the pSUPER vector. The shRNA against mouse GAPDH was obtained from EZbiolab. To generate luciferase reporter plasmids, the 3’UTR of Notch1 mRNA was amplified from genomic DNA by PCR and cloned into the pmirGLO vector (Promega) downstream of the Firefly Luciferase gene. The AU-rich element (141–367 of 3’UTR) was deleted to generate the Notch1 3’UTR-dARE plasmid. A Renilla Luciferase, serving as the internal control, is expressed independently from the same pmirGLO plasmid. The pCAG-mGFP (#14757), CBFRE-EGFP (#17705) and 3XFlagNICD1 (#20183) plasmids were obtained from Addgene. Myc-DDK-GAPDH was obtained from the Origene. Human GLO1 cloned into lentiviral vector pLX304, from ORFeome initiative, was acquired from DNASU. All clones were verified by sequencing. Recombinant rabbit GAPDH was purchased from Sigma.
Rodrigues D.C., Harvey E.M., Suraj R., Erickson S.L., Mohammad L., Ren M., Liu H., He G., Kaplan D.R., Ellis J, & Yang G. (2020). Methylglyoxal couples metabolic and translational control of Notch signalling in mammalian neural stem cells. Nature Communications, 11, 2018.
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5

Electroporation of Expression Vectors in Chick Neural Tube

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The following expression vectors were used as controls: pCAGG, pCAGGS-EGFP (Krispin et al., 2010b (link)), pCAG-mGFP (Addgene #14757), pCAGGS-RFP (Ofek et al., 2021 (link)). PCAGGS-RARα403 and PCAGGS-Cyp26A1 subcloned from PCAGGS-RARα403-IRES-eGFP and PCAGGS-Cyp26A1-IRES-eGFP, respectively, from S. Sockanathan (Novitch et al., 2003 (link)), were applied to inhibit RA activity. pCAB-cSmad6 was implemented to inhibit BMP activity (Nitzan et al., 2016 (link)). pCAG-VP16-RARα-IRES-eGFP was used to activate RA activity (Novitch et al., 2003 (link)).
To monitor RA activity, we used the pGL3-RARE-SV40-AP reporter [from J. Sen, Gupta and Sen, 2015 (link)]. Fluorescent versions of this reporter were prepared by replacing the AP tag with either RFP or with a destabilized version of GFP pGL3-RARE-SV40-RFP and pGL3-RARE-SV40-d2EGFP, respectively (Clontech). Wnt activity was measured with 12XTOPFLASH‐d2EGFP (Rios et al., 2010 (link)).
Unilateral, bilateral and/or double electroporations to the NT were performed as detailed under Results. To this end, DNA (4 ug/ul) was mixed with Fast Green and microinjected into the lumen of the NT at the flank level of the axis. Five mm tungsten electrodes were placed on either side of the embryo. A square wave electroporator (BTX, San Diego, CA, USA) was used to deliver one pulse of current at 17–25 V for 6ms.
Rekler D, & Kalcheim C. (2022). Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling. eLife, 11, e72723.
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6

Investigating SALL4 Regulation of ITGA6 and ITGB1

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The SALL4A and SALL4B coding regions were cloned downstream of the CAG promoter obtained from pCAG-mGFP (Addgene, 14757). To assess the promoter activities, 1kbp regions upstream of the transcription start sites of the ITGA6 and ITGB1 genes were linked to the minimal promoter in the pGL4.30 vector that also carries a firefly luciferase2 gene (Promega, E8481, Madison, WI, USA). The promoter reporter was co-transfected into Lenti-X 293T cells with the pGL4.73 vector that has the SV40 promoter and a Renilla luciferase gene (Promega, E6911), and with the SALL4 expression vector. A transfection reagent, FuGENE HD (Promega, E2311) was used.
One day after transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega, E2920).
Itou J., Tanaka S., Li W., Iida A., Sehara-Fujisawa A., Sato F, & Toi M. (2017). The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells. Biochimica et biophysica acta. Molecular cell research, 1864(1).
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