Lentiviral vectors carrying human precursor (pre) miR-15a (#
PMIRH15aPA-1), non-functional human GFP control miR (#
PMIRH000-PA-1), mouse pre-miR-15a (#MMIR-15a+16-1-PA-CL), and non-functional mouse GFP control miR (#
MMIR-000-PA-1) constructs were purchased from System Biosciences. Lentiviral particles were generated in HEK293T cells (3×10
6/8 mL 10% FCS DMEM medium) that were co-transfected with lentiviral miRNA vector (10 μg), and packaging plasmids (pVSV-G [5 μg], pMDL [10 μg], pREV [5 μg]) using polyethyleneimine (PEI) transfection reagent at a ratio of 1:4 for DNA and PEI for 24 h followed by fresh DMEM (10% FCS) media replacement. The lentiviral supernatants were collected at 48 and 96 h, centrifuged (200 g/5 min), and filtrated through a 0.45-μm syringe filter. The lentivirus was concentrated using a PEG-
it virus precipitation solution (System Biosciences,
LV825A-1). For viral infections, NB cells were seeded in a 6-well plate (1×10
5 (
link)/well) and were infected with lentivirus at 40% confluence for 24 h followed by fresh regular growth medium replacement, and the cells were allowed to grow for 3 to 5 days. The transduced cells were selected in the presence of puromycin (5 μg/mL). Further, to keep a pure population, GFP-positive cells were sorted by flow cytometry.
Pathania A.S., Prathipati P., Olwenyi O.A., Chava S., Smith O.V., Gupta S.C., Chaturvedi N.K., Byrareddy S.N., Coulter D.W, & Challagundla K.B. (2022). miR-15a and miR-15b modulate natural killer and CD8+T-cell activation and anti-tumor immune response by targeting PD-L1 in neuroblastoma. Molecular Therapy Oncolytics, 25, 308-329.
