Prrlsin cppt pgk gfp wpre lentiviral vector

DamID lentiviral constructs were a gift from Bas van Steensel and are previously described (Vogel et al., 2007 (link)). For rescue experiments, Tm7sf2 with a C-terminal EGFP tag was cloned by moving the gene from pEGFP-N2 (Malik et al., 2010 (link)) into pRRLSIN.cPPT.PGK-GFP.WPRE lentiviral vector (Addgene #12252). TM7SF2 point mutations were obtained from studies on C14SR homologs (Prakash and Kasbekar, 2002 (link); Li et al., 2015 (link)) and generated in the lentiviral vector by site-directed mutagenesis. All lentiviruses were then generated from these constructs using standard techniques as previously described (Gatticchi et al., 2019 (link)).

Lentiviral vectors for TCR expression were generated as previously described (56 (link)). Basically, the genes encoding TCR variable regions of TCR from single-cell V(D)J sequencing were fused with murine TCRα and β2 constant regions, respectively. The TCRα and β chains were linked by a P2A self-cleaving peptide. The TCR expression cassette was codon optimized, synthesized (GenScript, Nanjing, China), and cloned into the pRRLSIN.cPPT.PGK-GFP.WPRE lentiviral vector (Addgene, USA). TCR-encoding lentivirus were collected from 293T cells transfected with the transfer plasmid and the packaging plasmids PsPAX2, pMD2.G (Addgene, USA). 48 h and 72 h after transfection, cell supernatants were collected and filtered through 0.45 μm syringe filters (Sartorius, Germany). Virus particles were pelleted by ultracentrifugation (Beckman Coulter, USA), resuspended in T009, aliquoted on ice, and stored at -80°C.
To determine the viral infection titer, jurkat cells were infected with the lentivirus at different volumes, and the expression of exogenous TCR-β after transduction was detected by flow cytometry. The infection titer is calculated using the formula: Infection titer (IU/mL) = cell number at infection time × positive rate/virus volume.