Lentiviral Transduction of Retinal Ganglion Cells

Lentivirus was produced using calcium phosphate transfection as described74 (link). One day before transfection 5 × 10⁶ 293T cells (Clontech) were seeded onto a 10-cm cell culture plate. The following day cells were transfected with 15 μg MD2.G (Addgene #12259), 6 μg MDL/pRRE (Addgene #12251), 6 μg RSV/rev (Addgene #12253), and 15 μg of the GFP-transfer vector (Addgene #17451)75 (link). At 24 hours post transfection, 10 mM sodium butyrate was added to the cells. Lentivirus was harvested and concentrated at 48 hours post transfection using Lenti-X Concentrator (Clontech). To transduce sorted RGCs, lentivirus with 8 μg/mL Polybrene (Sigma) was added to the cell culture media overnight at 5–10 multiplicity of infection. The next day, the cells were washed with PBS and cultured with N2B27 media.