Lentiviral transduction for shRNA gene silencing and CRISPR/Cas9-mediated gene deletion

Lentiviral plasmids encoding shRNAs were obtained from Sigma-Aldrich and all-in-one vectors carrying CTNNB1 sgRNA/Cas9 with GFP reporter were obtained from Applied Biological Materials. Each plasmid was transformed into One Shot® Stbl3™ chemically competent cells (Invitrogen) and purified by ZymoPURE plasmid Maxiprep kit (Zymo research). Lentiviral pseudoparticles were obtained after plasmid transfection of 293FT cells using Lipofectamine 2000 (Invitrogen). The lentivirus-containing media was harvested 48 or 72 h after transfection and concentrated 40 – 50 times using Lenti-X concentrator (Takara Clontech). Sorted T_reg cells were stimulated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) for 24 h and transduced with lentiviral particles by spinfection (1000 x g for 90 min at 32°C) in the presence of Polybrene (5 μg/ml) on the plates coated with Retronectin (50 μg/ml) (Takara/Clontech) and anti-CD3 (1–2 μg/ml). Human Jurkat T cells were directly transduced with lentiviral particles by spinfection. Five days after transduction, cells were sorted on the basis of expression of GFP. GFP expressing human Jurkat T cells were further purified by FACS at least three times before using for experiments.