Puromycin was used on the selected monoclonal cell population after lentiviral particle transfection.
We obtained more than 95% lentiviral transduction efficiency of AD-MSCs according to the selection of
puromycin as a beneficial agent for ideal therapeutic purposes [68].
Twenty-four hours later,
puromycin (2 [micro]g/mL) was added.
Linearized targeting construct (10 [micro]g, TakaRa, Shiga, Japan) and Each TALEN expression plasmid (15[micro]g) were electroporated to passage one cells (5x[10.sup.6]), and then seeded in 6-well plates (1.5x 105 cells/well) and
puromycin selection (1 [micro]g/mL) was applied at 24h post-seeding.
The resulting virus-containing supernatant was then used to infect EML cells, and the positive cells were screened using the dual markers for GFP and
puromycin.
After 48 h incubation, antibiotic selection medium ([alpha]-MEM growth medium with 10 [micro]g/ml
puromycin) was used to kill all the untransduced cells.
After 48 h, transfectants were diluted in 1:10 ratio, and the antibiotic selection was performed in a medium containing 5 [micro]g/ml
puromycin (Life Technologies, USA) for 4 weeks.