Retronectin

pRV-luc plasmid was co-transfected in Plate-E with the packaging plasmid, pCL-ECO, using lipofectamine 2000 (Invitrogen, Carlsbad, CA). Supernatants were collected 48 and 72 h later and were combined to generate stocks, which were then used to transduce OT-I cells. Briefly, single-cell suspensions of OT-I cells were activated with Concanavalin A (2 μg/ml; Sigma, St. Louis, MO) and mouse IL7 (1ng/ml; PeproTech, Rocky Hill, NJ) for 24 h. Cells were then transduced with pRV-luc retrovirus in non–tissue culture 24-well plates precoated with RetroNectin (Takara Bio. Inc., Shiga, Japan). The transduced OT-I cells were then cultured for 48 hours in fresh medium supplemented with hIL-2 (300 U/ml; NIH AIDS Reagent Program, Germantown, MD) to allow the cells to recover. They were then used directly for adoptive transfer.

Retroviral particles were packaged by transient cotransfection of the Phoenix-Eco packaging cell line with the retroviral construct and the pCL-Eco plasmid (Imgenex) using FuGENE 6 (Promega). Viral supernatants were collected at 2 and 3 days after transfection and immediately frozen at −80°C until use. To infect BM-derived T-cell progenitors, 33 μg/mL retronectin (Clontech) and 2.67 μg/mL of DL1-extracellular domain fused to human IgG1 Fc protein (Varnum-Finney et al., 2000 (link)) were added in a volume of 500 μL per well in 24-well tissue culture plates (Costar, Corning) and incubated overnight. Viral supernatants were added next day into coated wells and spun down at 2000 rcf for 2 hr at room temperature. BM-derived T-cell progenitors used for viral transduction were cultured for 5 days according to conditions described above, disaggregated, filtered through a 40 μm nylon mesh, and 10⁶ cells transferred onto each retronectin/DL1-coated virus-bound 24-well supplemented with 5 ng/mL SCF (Peprotech), 5 ng/mL Flt3-L, and 5 ng/mL IL-7.