Generating Lentiviral Particles for Transduction

sfGFP plasmid^{51 (link)} gifted by David Thompson from the Church Lab was cloned into a pLenti CMV Puro DEST (Addgene, 17452) vector using LR Clonase II and transformed into DH5alpha cells (NEB, C2987H). Purified pLenti-sfGFP plasmid DNA (37.5 ug) was diluted into 1 ml Opti-MEM I (ThermoFisher, 319850672) with lentivirus pCMV-VSV-G envelope (Addgene, 8454) and psPAX2 packaging plasmid (Addgene, 12260) in a 3:3:1 mass ratio. FuGene6 (56.25 μl; Promega, E2691) was added to the mixture and incubated for 20 min at r.t. The transfection mix was then diluted in 15 ml antibiotic-free media consisting of Dulbecco’s modified Eagle medium (DMEM), high glucose (Gibco, 11965118), 10% fetal bovine serum, 0.1 mM minimal essential media non-essential amino acids (MEM-NEAA) (Gibco, 11140050), 6 mM l-glutamine, and 1 mM sodium pyruvate, and added to a T75 flask that was plated 1 d before transfection with 5.5 million human embryonic kidney (HEK) 293T cells. After 3 d, media with packaged lentivirus were collected and concentrated 50× using Lenti-X concentrator (Takara Bio, 631231), aliquoted and frozen at −80 °C. Single-cell clones of PGP1-SV NGN1 were subjected to transduction by packaged lentivirus for 1 d. Cells were grown until confluent, flow-sorted in a Sony SH800S cell sorter for the top 10% of sfGFP+ cells, expanded and frozen.