Soluble OVA gene, which was amplified from pCI-neo-sOVA (a gift from Maria Castro, Addgene plasmid 25 09818 (link)), and monomeric enhanced green fluorescent protein (mEGFP) gene, which was amplified from mEGFP-N1 (a kind gift from Michael Davidson, Addgene plasmid 54767), were genetically fused via P2A translational skipping sequence and were cloned in the Sleeping Beauty transposon plasmid with the human elongation factor 1α promoter.19 (link) This plasmid, pT2-EF-OVA-mEGFP, was electroporated together with the Sleeping Beauty Transposase plasmid, pCMV(CAT)T7-SB100 (a gift from Zsuzsanna Izsvak; Addgene plasmid #34 87920 (link)), into TRAMP-C2 by Nucleofector 4D instrument. The electroporated cells were kept in maintenance medium (DMEM supplemented with 10% FBS, 0.005 mg/mL bovine insulin, 1 nM DHT and cell sorted based on mEGFP expression using FACS Aria I cell sorter. The expression of OVA on sorted cells were confirmed by western blotting using rabbit polyclonal OVA antibody (ab186717) at 1:4000 dilution and flow cytometry using PE anti-mouse H-2Kb bound to SIINFEKL (BioLegend) before incubating with or without MCTP-39 for 48 hours. The expression of OVA was analyzed after 48 hours by flow cytometry using BDLSRIIA cytometer, and data were analyzed by FCS Express V.7 Research Edition.
Protocol detail
Engineered TRAMP-C2 Cells Express OVA and mEGFP
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Antibody
Aria
Bovine
Cells
Dilution
Elongation factor 1α
Enhanced green fluorescent protein
Flow cytometry
Gene
Human
Insulin
Mctp
Mouse
Ova protein
Plasmid
Rabbit
Translational
Transposase
Transposon
Corresponding Organization : Roswell Park Comprehensive Cancer Center
Other organizations : University of Leeds
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Variable analysis
independent variables
- Transfection of TRAMP-C2 cells with pT2-EF-OVA-mEGFP plasmid and pCMV(CAT)T7-SB100 plasmid
- Expression of OVA protein in sorted TRAMP-C2 cells
- Expression of mEGFP in sorted TRAMP-C2 cells
- Cytotoxicity of MCTP-39 on OVA-expressing TRAMP-C2 cells
- Maintenance medium (DMEM supplemented with 10% FBS, 0.005 mg/mL bovine insulin, 1 nM DHT)
- Incubation time of 48 hours with or without MCTP-39
- Untransfected TRAMP-C2 cells as a control for OVA and mEGFP expression
- Untreated TRAMP-C2 cells as a control for MCTP-39 cytotoxicity
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