Figure 1.

Overview of retrovirus production and transduction of activated primary murine T cells. (A) For retrovirus production, plated Phoenix Eco cells are transfected with Turbofect and plasmid mix. After 24 h the medium is refreshed, and at 48 and 72 h, the supernatants are collected, and the virus is concentrated by ultracentrifugation and directly used or frozen. (B) For the transduction of primary murine T cells, retrovirus is applied to plates precoated with retronectin and then spun. Activated T cells are then added to the plate, which is subsequently spun. At 48 h after transduction, the T cells are split and hIL-7/IL-15 added to the culture medium. At day 7 after transduction, CAR cell-surface expression is determined by flow cytometry, and the engineered T cells are evaluated in vitro and in vivo. Further details are provided in Materials and methods.