All lentiviruses were produced through transfection of human embryonic kidney cells expressing the SV40 large T-antigen (HEK293T).25 HEK293T cells were grown to approximately 50% confluence for calcium phosphate transfection in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Prior to preparation, the media was refreshed. Lentiviral envelope and capsid plasmids (VsVg, RRE, and Rev) were mixed with water and calcium (2.5 M) for a final volume of 0.5 ml. This solution was added dropwise to a 0.5 ml solution of 2× HEPES-buffered phosphate solution. The solution was incubated at room temperature in the dark for 30 minutes, and then added to the culture media. Using this procedure, the following lentiviruses were produced: reverse transcription transactivator (rtTa), transcription factors Ngn2 (with puromycin resistance), Dlx2 (with hygromycin resistance), Ascl1 (with puromycin resistance), hM3Dq-mCherry, tdTomato, and wild-type neuroligin-3 (NL3). Medium containing the packaged lentiviruses was collected on day 2 and 3 after transfection, aliquoted, and stored at −80 °C.
