Generation of Pseudo-Typed Lentiviral Particles

To generate pseudo-typed lentiviral particles, shRNA-encoding pLK4 vectors were transfected into 293 T cells along with pCL-Eco and Pax2 packaging vectors and VSV-G envelope vector. At 18–24 h prior to transfection, 7.5 × 10⁶ cells were seeded into one 15 cm tissue culture dish in cDMEM and incubated overnight. At 1–2 h prior to transfection, the media was replaced with fresh cDMEM. Lentiviral DNA was introduced into 293 T cells using the calcium phosphate transfection method. Per transfection, plasmids were combined at a ratio of 6:4:3 (30 ug lentiviral plasmid containing the desired construct, 20 ug packaging vectors, and 15 ug envelope vector) with CaCl₂ at a final concentration of 125 mM and HEPES-buffered saline at a final concentration of 50 mM HEPES pH 7.05, 140 mM NaCl, and 1.5 mM Na₂HPO₄). DNA was allowed to precipitate for 30 min at room temperature, then added drop-wise to 293 T cells. After 12–18 h, the transfection media was replaced with fresh cDMEM media combined with unsupplemented DMEM at a ratio of 20/80 for a final FBS concentration of 2%. Viral supernatant was harvested at 24 h intervals over the following 3 days, then spun down and filtered through 0.45-um Durapore Millex (Millipore) filters. Virus was diluted in a solution of 10% (w/v) PEG-8000 and 300 mM NaCl, rotated overnight at 4 °C, and spun down at 3000 g for 1 h at 4 °C. The viral pellet was held overnight at 4 °C in 1× PBS at 1/100th the original volume of the viral supernatant and spun down to pellet serum protein and other particulates. Concentrated virus was stored at -80 °C or used immediately to transduction. HUT 78 T lymphocytes at 0.5 × 10⁶ cells/mL were allowed to grow for 2–3 days in the presence of concentrated virus and hexadimethrine bromide (Polybrene) transfection reagent (Sigma Aldrich) at a concentration of 8 μg/mL before removal of the virus and initiation of puromycin selection. Cells were allowed to expand in the presence of increasing puromycin concentration, from 0.25 μg/mL to a final selection concentration of 1 μg/mL. Whole cell lysates were probed for loss of GRB2 expression and expression of GRB2 mutants after 2–3 passages in puromycin.