Packaging pcl eco plasmid

Mouse Pim2 cDNA was cloned into the MSCV-IRES-GFP plasmid. Empty or PIM-2–expressing retroviruses were produced in GP2-293 cells with pCL-Eco packaging plasmid (Addgene), using PEI MAX (Polysciences) according to the manufacturer’s instructions. BM for transduction was isolated by flushing femurs and tibias from Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice 4 days after treatment with 5 mg of 5-fluorouracil (Sigma-Aldrich). BM cells (2 × 10⁶/ml) were seeded in a 24-well plate in 1 ml of culture medium and 1 ml of retroviral supernatants supplemented with polybrene (10 μg/ml; EMD Millipore). Cells were spin-infected at 230g for 1.5 hours at 32°C and then incubated at 37°C for 4 to 6 hours. Transduced BM cells were washed twice and cultured in Iscove’s modified Dulbecco’s medium + 15% FBS + 1× penicillin/streptomycin + stem cell factor (50 ng/ml) + IL-3 (20 ng/ml) + IL-6 (10 ng/ml) (PeproTech). Three days later, cells were washed and transferred to a lethally irradiated CD45.1 congenic recipient. Eight weeks later, NK cells were isolated from the spleen for flow cytometry analysis.

To package retrovirus, 2.5x10⁶ HEK-293T cells were plated on a 10 cm dish in complete DMEM 18 hrs before transfection to produce ecotropic virus. The cells were transfected using the calcium phosphate transfection method by pre-mixing 10 μg of CAR or cytokine plasmid and 10 μg of pCL-Eco packaging plasmid (Addgene 12371) with 250 mM CaCl₂ in 510μL water prior to mixing with 510μL 2X HEPE-buffered saline (HBS; Alfa Aesar J62623) and dropwise addition to 4 x 10⁶ HEK 293T cells. Media was replaced with fresh media approximately 8 hrs post calcium phosphate treatment. Viral supernatants were harvested 48 hrs post transfection and filtered through a 0.45 μM filter before immediate use or flash freezing and storing at −80°C.

HEK 293T/17 cells (ATCC® CRL-11268, validated by ATCC) were used no longer than 4 weeks after thawing and were not tested for Mycoplasma. Retroviruses were prepared by co-transfecting HEK 293T/17 cells in a 10-cm plate with 10 μg of packaging pCL-ECO plasmid (Addgene #12371) and 10 μg MIGR1 based vectors by using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to manufacturer’s protocol. Lentiviruses were generated by co-transfection of HEK 293T/17 cells with 10 μg of pLVX-CMV-hTet2(CD)-flag-IRES-mRFP1 based vectors, and 10 μg packaging pCMV delta R8.2 plasmid (Addgene #12263) and envelope pVSV-G plasmid (Clontech, #PT3343-5). Viruses were harvested 40h and 64h after the transfection and filtered through a 0.45-μm PES filter (Millipore). For infection, 5 × 10⁵ cells were suspended in 1 ml of virus-containing medium with 6 μg/ml polybrene (Sigma). GFP+ and RFP+ cells were sorted 48 hrs after the initial infection.