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Pci neo cova

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The PCI-neo-cOVA is a plasmid vector designed for expressing the chicken ovalbumin (cOVA) gene. It contains a neomycin resistance gene for selection in mammalian cells.

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Lab products found in correlation

Pslik hygro, by Addgene (2 mentions) Tmem192, by OriGene (2 mentions) Hla a, by Addgene (2 mentions) Pbabe zeo, by Addgene (2 mentions) Pbabe cova, by Addgene (2 mentions) Gateway cloning system, by Thermo Fisher Scientific (2 mentions) Pinducer20, by Addgene (2 mentions) Nebuilder hifi dna assembly cloning kit, by New England Biolabs (2 mentions) Matrigel, by BD (1 mentions) Pci neo mova, by Addgene (1 mentions) Pcdna3 ova, by Addgene (1 mentions) Pci neo sova, by Addgene (1 mentions) Antibiotic antimycotic aa, by Thermo Fisher Scientific (1 mentions) Genehogs, by Thermo Fisher Scientific (1 mentions) Pcmv egfp, by Addgene (1 mentions) Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Pureyield plasmid midiprep system, by Promega (1 mentions)

5 protocols using pci neo cova

1

Murine Pancreatic Tumor Model Establishment

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The “FC1242” (C57BL/6 background) murine PDA cell line was isolated from the pancreatic tumor of a KPC mouse (pdx1cre/+;KRasLSL-G12D/+; p53R172H/+) as we described previously51 (link). In select experiments, we utilized FC1242 KPC-derived tumor cells (1 × 106), which we engineered to express OVA using pCI-neo-cOVA (gift of Maria Castro; Addgene plasmid # 25097) as we described21 (link). To establish orthotopic pancreatic lesions in vivo, tumor cells (105) were suspended in phosphate-buffered saline (PBS) with Matrigel (BD) in a 1:1 ratio and were injected into the body of the pancreas via laparotomy. In select experiments, DC (2.5 × 104) were admixed with tumor cells and co-transferred to pancreata of mice. In other experiments, OTII T cells (5 × 104) were admixed with equal numbers of FC1242 and FC1242.Ova tumor cells (2.5 × 104 each) and administered subcutaneously. For our liver metastases model, PDA cells (1 × 106) were administered into the portal venous system via direct splenic injection followed by splenectomy, as we reported52 (link).
Barilla R.M., Diskin B., Caso R.C., Lee K.B., Mohan N., Buttar C., Adam S., Sekendiz Z., Wang J., Salas R.D., Cassini M.F., Karlen J., Sundberg B., Akbar H., Levchenko D., Gakhal I., Gutierrez J., Wang W., Hundeyin M., Torres-Hernandez A., Leinwand J., Kurz E., Rossi J.A., Mishra A., Liria M., Sanchez G., Panta J., Loke P., Aykut B, & Miller G. (2019). Specialized dendritic cells induce tumor-promoting IL-10+IL-17+ FoxP3neg regulatory CD4+ T cells in pancreatic carcinoma. Nature Communications, 10, 1424.
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2

Generation of Fluorescent Protein Constructs

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GFP-NBR1 and GFP-NBR1 dUBA were provided by Jayanta Debnath. LysoTag - TMEM192-mRFP-3xHA (TMRHA) - was generated by subcloning the cDNA of TMEM192 (Origene) together with monomeric Red Fluorescent Protein (mRFP) and 3xHA tag into the NheI and EcoRI sites of pLJM1 lentiviral vector. HLA-A-TurboID-FLAG (HLA-A-TrID) was generated by subcloning the cDNA of HLA-A (Addgene plasmid, #85162) into the EcoRI and NotI sites of the TurboID pLVX vector (gift from Roberto Zoncu, UC Berkeley). pMXs GFP-LC3-RFP was a gift from Noboru Mizushima (Addgene, plasmid #117413). For Dox-inducible expression of Atg4BC74A, mTurquoise2 or mStrawberry was fused to Atg4BC74A and inserted into either pSLIK-Hygro (used for in vitro studies) (Addgene, plasmid #25737) or pINDUCER20 (used for in vivo studies) (Addgene, plasmid #44012), using the Gateway Cloning system (Thermo Fisher Science). For the generation of OVA-expressing cells, the cOVA fragment was cloned from pCI-neo-cOVA (Addgene, plasmid #25097), fused with 2A peptide and mStrawberry sequences using NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) according to manufacturer’s instruction, and inserted into the EcoRI and SalI sites of pBabe-zeo (Addgene, plasmid #1766) to generate pBabe-cOVA-2A-mStrawberry. Stable cOVA expression was confirmed and monitored with mStrawberry fluorescence.
Yamamoto K., Venida A., Yano J., Biancur D.E., Kakiuchi M., Gupta S., Sohn A.S., Mukhopadhyay S., Lin E.Y., Parker S.J., Banh R.S., Paulo J.A., Wen K.W., Debnath J., Kim G.E., Mancias J.D., Fearon D.T., Perera R.M, & Kimmelman A.C. (2020). Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature, 581(7806), 100-105.
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3

Generating Ovalbumin-Expressing Lentiviral Vectors

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Plasmids (pCI-neo-sOVA, pCI-neo-mOVA, pCI-neo-cOVA, pcDNA3-OVA) were obtained from Addgene. Different forms of artificial tumor antigen ovalbumin sequence were subcloned into a lentiCRISPR-V2-blast vector via Gibson assembly to generate different Ova-expressing vectors (lentiV2-blast-sOva, lentiV2-blast-mOva, lentiV2-blast-cOva, lentiV2-blast-Ova).
Wang X., Tokheim C., Gu S.S., Wang B., Tang Q., Li Y., Traugh N., Zeng Z., Zhang Y., Li Z., Zhang B., Fu J., Xiao T., Li W., Meyer C.A., Chu J., Jiang P., Cejas P., Lim K., Long H., Brown M, & Liu X.S. (2021). In Vivo CRISPR Screens Identify E3 Ligase Cop1 as a Modulator of Macrophage Infiltration and Cancer Immunotherapy Target. Cell, 184(21), 5357-5374.e22.
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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

Generation of Fluorescent Protein Constructs

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GFP-NBR1 and GFP-NBR1 dUBA were provided by Jayanta Debnath. LysoTag - TMEM192-mRFP-3xHA (TMRHA) - was generated by subcloning the cDNA of TMEM192 (Origene) together with monomeric Red Fluorescent Protein (mRFP) and 3xHA tag into the NheI and EcoRI sites of pLJM1 lentiviral vector. HLA-A-TurboID-FLAG (HLA-A-TrID) was generated by subcloning the cDNA of HLA-A (Addgene plasmid, #85162) into the EcoRI and NotI sites of the TurboID pLVX vector (gift from Roberto Zoncu, UC Berkeley). pMXs GFP-LC3-RFP was a gift from Noboru Mizushima (Addgene, plasmid #117413). For Dox-inducible expression of Atg4BC74A, mTurquoise2 or mStrawberry was fused to Atg4BC74A and inserted into either pSLIK-Hygro (used for in vitro studies) (Addgene, plasmid #25737) or pINDUCER20 (used for in vivo studies) (Addgene, plasmid #44012), using the Gateway Cloning system (Thermo Fisher Science). For the generation of OVA-expressing cells, the cOVA fragment was cloned from pCI-neo-cOVA (Addgene, plasmid #25097), fused with 2A peptide and mStrawberry sequences using NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) according to manufacturer’s instruction, and inserted into the EcoRI and SalI sites of pBabe-zeo (Addgene, plasmid #1766) to generate pBabe-cOVA-2A-mStrawberry. Stable cOVA expression was confirmed and monitored with mStrawberry fluorescence.
Yamamoto K., Venida A., Yano J., Biancur D.E., Kakiuchi M., Gupta S., Sohn A.S., Mukhopadhyay S., Lin E.Y., Parker S.J., Banh R.S., Paulo J.A., Wen K.W., Debnath J., Kim G.E., Mancias J.D., Fearon D.T., Perera R.M, & Kimmelman A.C. (2020). Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature, 581(7806), 100-105.
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