Pcmv6 ac gfp

Manufactured by OriGene

Sourced in United States

PCMV6-AC-GFP is a plasmid vector that expresses the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. The plasmid backbone contains an ampicillin resistance gene for selection in bacteria.

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Close spelling variants of this product

PCMV6-AC-GFP vector backbone PCMV6-AC-GFP empty vector control PCMV6-AC-GFP mammalian expression vector PCMV6‐AC‐GFP‐SIM2 PCMV6–AC–TRIM3–GFP TrueORF clone PCMV6-AC-GFP-ID1 (RG202061) PCMV6-AC-TDP-43-Gfp PCMV6-AC-GFP-Survivin PCMV6-AC-GFP without Insert PCMV6-AC-GFP vector

66 protocols using pcmv6 ac gfp

Plasmid Constructs for Studying Neurodegenerative Diseases

Cited 2 times

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M337V human TDP-43 cDNA was cloned into pCMV6-AC-GFP (Origene) to generate a mutant TDP-43 construct C-terminally tagged with tGFP18 (link). HA-NFAT1(1-460)-GFP was a gift from Anjana Rao (Addgene plasmid #11107). Huntingtin protein-encoding constructs, pT-Rex-Htt46Q-Tc1-mCherry and pT-Rex-Htt25Q-Tc1-mCherry, were gifts from Dr. Danny Hatters (University of Melbourne, Australia)19 (link). pEGFP-SOD1, pEGFP-SOD1-A4V, pEGFP-SOD1-G93A were gifts from Dr. Brad Turner (The Florey Institute of Neuroscience and Mental Health, Australia)20 (link). SOD1-tdTomato constructs were created by replacing the GFP sequences in the SOD1-GFP plasmids with tdTomato (Genscript)18 (link). The expression vector pCMV6-AC-GFP containing FUS was obtained from Origene and site directed mutagenesis was performed by Genscript to create the R495X mutant18 (link). Plasmids containing sequences encoding wild type (WT) and temperature-sensitive double mutant (R188Q, R261Q; DM) firefly luciferase-eGFP were a gift from Prof Ulrich Hartl (Max Plank Institute, Germany)21 and were recloned into pcDNA4/TO (Life Technologies) for mammalian cell transfection.

Whiten D.R., San Gil R., McAlary L., Yerbury J.J., Ecroyd H, & Wilson M.R. (2016). Rapid flow cytometric measurement of protein inclusions and nuclear trafficking. Scientific Reports, 6, 31138.

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Transfection of NSCs with SOX21 Vector

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Lipofectamine 3000 reagent (Thermo Fisher Scientific, MA, USA) was used to transfect NSCs with mammalian vector pCMV6-AC-GFP containing SOX21 (NM_177753) Mouse Tagged ORF Clone (No: MG223510) or blank control pCMV6-AC-GFP (No: PS100010, OriGene Technologies, Inc. Rockville, MD, USA), according to the manufacturer’s instructions.

Wang H., Ma Z.W., Ho F.M., Sethi G, & Tang F.R. (2023). Dual Effects of miR-181b-2-3p/SOX21 Interaction on Microglia and Neural Stem Cells after Gamma Irradiation. Cells, 12(4), 649.

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AAV-Mediated Overexpression of Scf and Ptbp1 in Mice

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The AAV delivery system was used to overexpress soluble Scf-aequorea coerulescens GFP (acGFP) or Ptbp1^T138E-acGFP or acGFP in mouse. We first amplified the CMV-acGFP cassette from pCMV6-AC-GFP (Origene, #PS100010) and cloned it into pAAV.CMV.PI.GFP.WPRE.bGH (Addgene, #105530) by replacing the CMV-chimeric intron-EGFP following digestion with NheI and HindIII. Then we cloned EC-specific promoter of Flt-1 (2748 to 284) into this construct by replacing CMV promoter with NheI and SalI restriction enzymes according to a previous report^{48 (link)} to create the construct pAAV-Flt1-acGFP. The open reading frame (ORF) encoding soluble Scf or Ptbp1, without a stop codon, was cloned into pAAV-Flt1-acGFP following digestion with SalI and MluI to create pAAV-Flt1-sScf-acGFP or pAAV-Flt1-Ptbp1-acGFP, respectively. Vector pAAV-Flt1-acGFP served as a control. The AAV2 vector was generated by transfecting HEK293T cells with three plasmids (pAAV-Flt1-derived plasmid, AAV2 rep and cap genes plasmid, and the pAd helper plasmid). Titers of vector DNA were measured by quantitative PCR with vector-specific primers. Mice were injected with 100 μl virus containing 10¹¹ AAV2 vector genomes via the tail vein.

Zhao Q., Han Y.M., Song P., Liu Z., Yuan Z, & Zou M.H. (2022). Endothelial cell-specific expression of serine/threonine kinase 11 modulates dendritic cell differentiation. Nature Communications, 13, 648.

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HeLa Cells Transfection and Analysis

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HeLa Kyoto cells were grown in 6-well culture dishes for 24 h in complete medium at 37°C in 5% CO₂. Afterward, cells were transfected with a human RNF11 cDNA clone carboxy-terminally linked to turboGFP (pCMV6-AC-GFP; OriGene) using FuGENE6 (Roche) according to the manufacturer’s instructions. 24 h after transfection, cells were lysed and used for Q-RT-PCR experiments or fixed and immunostained.

Scharaw S., Iskar M., Ori A., Boncompain G., Laketa V., Poser I., Lundberg E., Perez F., Beck M., Bork P, & Pepperkok R. (2016). The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR. The Journal of Cell Biology, 215(4), 543-558.

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Plasmid Propagation and Subcloning Protocol

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Plasmids were propagated in E. coli, extracted and purified using a Labopass plasmid DNA purification kit (Cosmo Genetech, Seoul, Korea). Full-length human ERGIC3 (GenBank ID:NM_015966.2) was subcloned in pCMV6-AC-GFP (OriGene Technologies Inc., Rockville, MD, USA), pCMV-Myc (Clontech Laboratories Inc., Palo Alto, CA, USA) or pGBKT7 (Clontech) plasmid vectors. ERGIC3 shRNAs were purchased from OriGene.

Hong S.H., Chang S.H., Cho K.C., Kim S., Park S., Lee A.Y., Jiang H.L., Kim H.J., Lee S., Yu K.N., Seo H.W., Chae C., Kim K.P., Park J, & Cho M.H. (2016). Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy. Oncotarget, 7(40), 65335-65347.

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Overexpression of MSI2 in Breast Cancer

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A total of 5 μg MSI2-GFP and GFP vectors (pCMV6-MSI2-AC-GFP and pCMV6-AC-GFP from Origene, Rockville, MD, USA) were transfected into MCF7 and MDA-MB-231 cells (source: ATCC) using endofectin transfection reagent (Genocopoeia, Rockville, MD, USA) according to the manufacturer’s instructions. Expression was determined by fluorescence microscopy, western immunoblotting and qRT-PCR. Cell lines were authenticated and quality controlled at ATCC.

Abdalla M., Tran-Thanh D., Moreno J., Iakovlev V., Nair R., Kanwar N., Abdalla M., Lee J.P., Kwan J.Y., Cawthorn T.R., Warren K., Arneson N., Wang D.Y., Fox N.S., Youngson B.J., Miller N.A., Easson A.M., McCready D., Leong W.L., Boutros P.C, & Done S.J. (2017). Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma. Nature Communications, 8, 1245.

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Generating SLC25A1 Mutant Constructs

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The coding sequence of the SLC25A1 gene was recloned from pCMV6-AC-GFP (Origene, Rockville, MD, USA) into the pEGFP-N1 vector (Clontech). Subsequently, the enhanced green fluorescent protein (EGFP) was removed from the vector, as it interfered with protein function. For each of the 17 missense mutations included in this study, recombinant plasmids were generated by site-directed mutagenesis, as previously described (Betsalel et al. 2012 (link)). Successful mutagenesis and absence of PCR artifacts was confirmed by full-length sequencing of the SLC25A1 coding sequence.

Pop A., Williams M., Struys E.A., Monné M., Jansen E.E., De Grassi A., Kanhai W.A., Scarcia P., Ojeda M.R., Porcelli V., van Dooren S.J., Lennertz P., Nota B., Abdenur J.E., Coman D., Das A.M., El-Gharbawy A., Nuoffer J.M., Polic B., Santer R., Weinhold N., Zuccarelli B., Palmieri F., Palmieri L, & Salomons G.S. (2017). An overview of combined D-2- and L-2-hydroxyglutaric aciduria: functional analysis of CIC variants. Journal of Inherited Metabolic Disease, 41(2), 169-180.

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Transfection of MBOAT7 Plasmid in MDMs

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MBOAT7 plasmid (pCMV6-AC-MBOAT7-GFP, OriGene) and its empty cloning vector pCMV6-ACGFP, OriGene) were transfected into MDMs using a Nucleofector kit (Amaxa) as per manufacturer’s instructions for 48 h. Small interfering RNA (siRNA) transfection was performed with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions using 50 nM of the specific siRNA or non-targeting control for 48 h.

Alharthi J., Bayoumi A., Thabet K., Pan Z., Gloss B.S., Latchoumanin O., Lundberg M., Twine N.A., McLeod D., Alenizi S., Adams L.A., Weltman M., Berg T., Liddle C., George J, & Eslam M. (2022). A metabolic associated fatty liver disease risk variant in MBOAT7 regulates toll like receptor induced outcomes. Nature Communications, 13, 7430.

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Generation of Protein Expression Constructs

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The pEGFP-N1 vectors containing human SOD1^wt, SOD1^A4V and G93A SOD1 were generated as described65 (link). Expression vector pCMV6-AC-GFP containing human TDP-43 and FUS was obtained from OriGene (USA). SOD1-tomato constructs were generated by replacing the GFP sequences in the GFP-tagged constructs65 (link) with the tomato red fluorescent protein. TDP-43^wt-tomato (Addgene plasmid 28205, provided by Zuoshang Xu34 (link)), mRFP-Ubiquitin (Addgene plasmid 11935, provided by Nico Dantuma66 (link)) and pmRFP-LC3 (Addgene plasmid 21075, Provided by Tamotsu Yoshimori67 (link)) constructs were acquired from Addgene (USA). The vectors describing Httex1(46Q) fusions (C-terminally) to mCherry and Cerulean were generated as described33 (link)68 (link).

Farrawell N.E., Lambert-Smith I.A., Warraich S.T., Blair I.P., Saunders D.N., Hatters D.M, & Yerbury J.J. (2015). Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions. Scientific Reports, 5, 13416.

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Overexpression of OSM and CNTF in Breast Cancer Cells

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MCF7 cells with empty vector and OSM/CNTF overexpression were established
by transduction using these expression plasmids: pCMV3-C-GFPSpark Vector (Sino
Biologcial, Catalog Number CV026), pCMV3-C-OSM-GFP (Sino Biologcial, Catalog
Number HG10452-ACG), pCMV6-AC-GFP (Origene, Catalog Number PS100010) and
pCMV6-AC-CNTF-GFP (Origene, Catalog Number RG222331). Cells were selected using
hygromycin (OSM) and neomycin (CNTF). MDA-MB-231 parental and bone metastatic
cells transiently overexpressing OSM and CNTF were established using previously
mentioned expression vectors. Cells were transfected using Lipofectamine LTX
with Plus Reagent (Thermo Fisher, Catalog Number 15338030) and harvested 36
hours later.

Omokehinde T., Jotte A, & Johnson R.W. (2021). gp130 cytokines activate novel signaling pathways and alter bone dissemination in ER+ breast cancer cells. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 37(2), 185-201.

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