Primary T lymphocytes from healthy donors’ PB mononuclear cells were isolated and activated using magnetic beads conjugated to anti-human CD3 and CD28 antibodies (Dynabeads human T-activator CD3/CD28; Invitrogen) in IMDM medium (GIBCO-BRL) supplemented with penicillin, streptomycin, glutamine, 10% FBS, and 5 ng/ml of IL-7 and IL-15 (PeproTech) as described (42). After 2 days of stimulation, T cells were infected with the indicated IDLVs at MOI of 100. The following day, transduced T cells were electroporated with mRNAs encoding for ZFNs (106 cells/condition, 100 µg/ml of ZFNs; P3 Primary Cell 4D-Nucleofector X Kit, program EO 115; Lonza) and then expanded to perform flow cytometry and molecular analyses.
For CRISPR/Cas9 editing in human CD34+ cells (from Lonza), 2.5 µM of ribonucleoproteins (RNP) were electroporated. RNPs were made by incubating Cas9 protein (Integrated DNA Technologies) with synthetic 2’O-methyl-phosphorotyoate modified (10) sgRNA (Metabion, HPLC purified, intron 1 IL2R Gguide 8) at 1:1.5 molar ratio for 10 min at 25°C. For large scale experiments, electroporation was performed using Lonza 4D-Nucleofector LV Unit (P3 primary cell nucleofectorkit, program EO 100, Lonza).