Pmxs ires gfp retroviral vector

T-cell lines were single-cell sorted into 96-well PCR plates by using a cell sorter (BD FACSAria II, BD Biosciences), and were stored at −80°C until the RT-PCR reaction. The TCRα and β cDNAs were amplified by single-cell RT-PCR, and the PCR products were examined by direct sequencing. The TCR repertoire was analyzed with the IMGT/V-Quest tool (https://www.imgt.org/). The TCRβ and α cDNAs were linked with a self-cleaving P2A fragment, and inserted into the pMXs − IRES − GFP retroviral vector (Cell Biolabs) by using Gibson Assembly Master Mix (New England Biolabs), according to the manufacturer’s instructions. The nucleotide sequence of the plasmid DNA was confirmed by DNA sequencing. The constructed plasmid vector, pMXs/TCRβ−P2A − TCRα/−IRES − GFP, was then transfected into Phoenix A cells (kindly provided by Dr Garry Nolan, Stanford University) with FuGENE 6 (Roche). The culture supernatant was collected 72 h after the transfection. Human CD4⁺ TG40 cells were infected with the recombinant retroviruses by using retronectin (Takara Bio), according to the manufacturer’s instructions.

Lentiviral shRNA construct targeting FOXP1 was purchased from Sigma–Aldrich (Louis, MO). For generation of lentiviral particles, 293FT cells were transfected with the 6.67 μg targeted viral plasmid and lentiviral packaging mix; 5 μg of VSV-G and 3.33 μg of Δ8.9 using 15 μL Lipofectamine/Lipofectamine Plus reagent. For retroviral overexpression of FOXP1, FOXP1 cDNA was subcloned into BamHI and XhoI sites of the pMXs IRES GFP retroviral vector (#RTV-013; Cell Biolabs, Inc.) and then transfected with the 5 μg targeted viral plasmid, 5 μg of VSV and 5 μg of gag/pol into 293FT cells by using Lipofectamine/Lipofectamine Plus reagent. The culture supernatants containing lentivirus or retrovirus were harvested and concentrated using the Lenti-X Concentrator (#631231; Clontech) and Retro-X Concentrator (#631445; Clontech) at 48 h after transfection. For lentiviral or retroviral transduction, A2780 cells were infected with shRNA-bearing lentivirus or pMXs-RNA-bearing retrovirus in the presence of 5 μg/mL polybrene (Sigma-Aldrich). FOXP1-silenced or –overexpressed cells were selected for 10 days with 1μg/mL puromycin and then maintained in RPMI1640 supplemented with 10% FBS and 1 μg/mL puromycin.