4T1 and YAC-1 cells were purchased from the ATCC. E0771 cells were purchased from CH3 Biosystems. AT3 cell line was a generous gift from S.I. Abrams at Roswell Park Comprehensive Cancer Center. To generate AT3-Luc, E0771-Luc or 4T1-Luc cells, the tumor cells were infected with luciferase-expressing lentivirus (Addgene) (Campeau et al., 2009 (link)). For OVA labeling, the AT3-Luc cells were transfected with pcDNA3-OVA (Addgene) (Diebold et al., 2001 (link)) to generate AT3-OVA-Luc tumor cells. For mCherry labeling, AT3 cells were infected with mCherry-expressing lentivirus (Addgene) to generate AT3-mCherry cells. To overexpress G-CSF, AT3 cells were infected with g-csf (Csf3)-expressing lentivirus (the vector was a gift from R.A. Weinberg, Massachusetts Institute of Technology) to generate AT3-gcsf cells. To generate 4T1-GFP, AT3-GFP, AT3-gcsf-GFP and E0771-GFP cells, the tumor cells were infected with green fluorescent protein (GFP)-expressing lentivirus (Addgene). Human lung fibroblast-like cells (mesenchymal stem cells) were purchased from ScienCell Research Laboratories. All cell lines were maintained in a humidified 5% CO2 incubator at 37 °C and confirmed to be mycoplasma free.
Pcdna3 ova
Manufactured by Addgene
Sourced in United States
pCDNA3-OVA is a plasmid vector that contains the gene encoding ovalbumin (OVA), a widely used model antigen. This plasmid can be used for the expression of OVA in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Phiv luc zsgreen backbone, by Addgene (2 mentions)
Pmscv ires mcherry fp, by Addgene (2 mentions)
E0771, by Addgene (1 mentions)
Mcherry expressing lentivirus, by Addgene (1 mentions)
Yac 1 cells, by American Type Culture Collection (1 mentions)
Pci neo mova, by Addgene (1 mentions)
Pci neo sova, by Addgene (1 mentions)
Pci neo cova, by Addgene (1 mentions)
Bamhi, by Enzynomics (1 mentions)
Plv ef1a ires puro, by Addgene (1 mentions)
Pmdlg prre, by Addgene (1 mentions)
Protamine sulfate, by Merck Group (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Prsv rev, by Addgene (1 mentions)
8 protocols using pcdna3 ova
1
Generation and Characterization of Tumor Cell Lines
2
Generating Ovalbumin-Expressing Lentiviral Vectors
3
Generating H-2Kb-Ctag Fusion Construct
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The H-2Kb sequence was sub-cloned into cetHS-puro plasmid. As a result, Ctag sequence was fused to the C-terminus of the H-2Kb sequence. The successful generation of the construct was determined by PCR and sequencing (data not shown). One day prior to transfection the HEK293T cells were seeded in 10 cm tissue culture dish. By next day the cells reached 70–80% confluence. At this time, the culture medium was replaced with 9 mL DMEM medium containing 25 μM chloroquine and the cells transfected with plasmids coding for Kb-Ctag and OVA (pcDNA3-OVA; Addgene, plasmid #64599). Briefly, 5 μg of Kb-Ctag and 5 μg OVA expressing plasmids were mixed in 450 μL H2O in 1.5 mL Eppendorf tube; 500 μL 2X HBSS was added sequentially. 50 μL 2 M CaCl2 solution was then added and the tube was vortexed and kept on ice for 15 minutes. The plasmids were gently added on top of the cell cultures. For single transfections 10 μg of Kb-Ctag plasmid was used. On day 2 post transfection the cells were washed with warm DMEM medium twice and cultured for one extra day.
4
Generating Stable OVA-Expressing Tumor Cells
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OVA DNA (pcDNA3-OVA; Addgene, Watertown, MA, USA) was amplified with the following primers: 5′-AAT TGG ATC CTT AAG CTT GCC ACC ATG G-3′ and 5′-TTA TGA ATT CCT CGA GTT AAG GGG AAA CAC ATC TGC C-3′. OVA-encoding DNA fragments were digested with BamHI and EcoRI (Enzynomics, Daejeon, Korea) and inserted into the third-generation lentiviral plasmid pLV-EF1a-IRES-Puro (Addgene).
For assemble of lentivirus, Lenti-X™ 293 T cells were transfected with the OVA-encoding pLV plasmid and packaging plasmids (pRSV-Rev, pMDLg/pRRE, and pMD2.G; Addgene) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Hampton, NH, USA). The culture supernatant of transfected cells was filtered with 0.45 μm cellulose acetate filters (Sartorius, Göttingen, Germany).
GL261 tumor cells were spinoculated with OVA-encoding lentivirus and protamine sulfate (Sigma–Aldrich) by centrifugation at 1000 × g and 32 °C for 1 h. Transduced cells were selected with puromycin. To make single clones of GL261-OVA, transduced cells were diluted, plated at 0.5 cells/well, and incubated. Cells from wells containing a single colony were harvested, and OVA expression was confirmed with FITC-conjugated anti-OVA antibody (Abcam, Cambridge, United Kingdom) via flow cytometry.
For assemble of lentivirus, Lenti-X™ 293 T cells were transfected with the OVA-encoding pLV plasmid and packaging plasmids (pRSV-Rev, pMDLg/pRRE, and pMD2.G; Addgene) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Hampton, NH, USA). The culture supernatant of transfected cells was filtered with 0.45 μm cellulose acetate filters (Sartorius, Göttingen, Germany).
GL261 tumor cells were spinoculated with OVA-encoding lentivirus and protamine sulfate (Sigma–Aldrich) by centrifugation at 1000 × g and 32 °C for 1 h. Transduced cells were selected with puromycin. To make single clones of GL261-OVA, transduced cells were diluted, plated at 0.5 cells/well, and incubated. Cells from wells containing a single colony were harvested, and OVA expression was confirmed with FITC-conjugated anti-OVA antibody (Abcam, Cambridge, United Kingdom) via flow cytometry.
5
Lentiviral Constructs for Ovalbumin Expression
6
Lentiviral Constructs for Ovalbumin Expression
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pLenti6-UbC-VKine-HA and pLenti6-UbC-VKine-Myc has been previously described (Hope et al., 2016 (link); McCulloch et al., 2009 (link)). Ovalbumin (OVA) amplicon was PCR amplified from pcDNA3-OVA (Addgene #64599) and cloned into pHIV-Luc-ZsGreen backbone (Addgene #39196). All lentiviral constructs were transformed into NEB 5-alpha competent cells (#C2987U) for propagation of plasmid DNA. All plasmids were prepped and purified using Macharey-Nagel NucleoBond Xtra Maxi kit (# 740414.50).
7
Plasmid Construct Generation for Cellular Studies
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pBABE hygro MEN1 WT was a gift from Matthew Meyerson (Addgene 11024)85 (link). pCDH-EF1-Puro lentiviral vectors encoding HA-FLAG-tagged wildtype H3.3 and H3.3 K27M were a kind gift from Peter Lewis (University of Wisconsin)86 . All plasmids were verified by Sanger sequencing analysis through the Australian Genome Research Facility (Melbourne, Victoria). An ovalbumin (OVA) expressing retroviral vector was generated by PCR amplification of full-length chicken OVA from pcDNA3-OVA (Addgene 64599, a gift from Sandra Diebold & Martin Zenke)87 (link) and cloned into both pMSCV-IRES-mCherry FP (a gift from Paul Beavis) and pMSCV-IRES-GFP (modified from pMSCV-IRES-mCherry FP) via EcoRI and XhoI sites.
8
Plasmid Construct Generation for Cellular Studies
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