Pcdh ef1 fhc

pCDNA3-Flip-GFP (Casp3 cleavage seq) T2A mCherry was a gift from Xiaokun Shu (Addgene plasmid #124428) (Zhang et al., 2019 (link)). To prevent silencing and improve stability of high-level expression, the construct was re-cloned into pCDH-EF1-FHC, a gift from Richard Wood (Addgene plasmid #64874), in order to have the EF-1α promoter driving the construct (Wang et al., 2017 (link)).
293T cells were plated in antibiotic-free DMEM medium. Plasmids used were pMD2G (0.3 μg), pPAX2 (2.7 μg), and EF1α -mCherry-Flip-GFP (3 μg). Transfection was performed with Lipofectamine 2000. 16 hours later, the medium was aspirated and replaced with bovine serum albumin (BSA) enriched medium. The following day, this medium was collected, filtered and applied to BHT101 and OCUT2 cell lines in the presence of 0.8 μg/ml polybrene The next day the medium was aspirated and replaced with fresh medium. Puromycin selection was initiated with 1.5 μg/mL. After several days of selection, cells were FACS-sorted to select the population expressing the highest levels of mCherry. Cell lines were expanded in the presence of 1.5 μg/mL Puromycin.

We subcloned mouse NRP1 from pCherry-mNrp1 (Addgene, cat#21934) into the
cloning site of the lentivirus vector pCDH-EF1-FHC (Addgene, cat#64874) and then
produced lentivirus using the packaging plasmids psPAX2 (Addgene, cat#12260),
and pMD2.G (cat#12259). Then SA iTreg were infected by the NRP1 lentivirus, and
the function of these iTreg was detected by performing the cytotoxicity
assay.