Figure 3
From: Optimized conditions for gene transduction into primary immune cells using viral vectors
(A,B) T- or NK cells were activated by co-culture of PBMCs with CD3/CD28 dynabeads or aAPCs, respectively. After 3 days, RDF pseudotyped retroviral luciferase genes were transduced into T-cells (A) or NK cells (B) under different polybrene concentrations. Luminescence and cell numbers were measured 48 h after gene transduction. The 1 × represents 40 µg/ml of polybrene. Gray bars or orange graph indicates luminescence or relative cell number, respectively. (C) NK cells were transduced with RDF-pseudotyped retroviral GFP genes with or without polybrene and spinoculation. After 1 week, FACS analysis was performed. (D) 293T-cells were infected with different volumes of VSVg-pseudotyped luciferase lentiviruses. After 4 days, luminescence was measured. (E) 293 T-cells were infected with different concentration of VSVg-pseudotyped luciferase lentiviral particles. After 5 days, luminescence was measured. (F) PBMCs were cultured with CD3/CD28 dynabeads to proliferate T-cells. After 7 days (7d), VSVg-pseudotyped luciferase lentiviral particles were infected into activated T-cells at different concentrations of viral particles and at different g forces. After 4 days (11d), luminescence was measured. (G) Different numbers of T-cells were infected with VSVg-pseudotyped luciferase lentiviruses, whose RNA copy number was 1.7 × 1010. After 3 days, luminescence was measured.
