Lentiviral vector plasmids carrying scrambled short hairpin RNA (scr-sh), MeCP2 shRNA and supporting plasmids (Vsvg, del 8.9) were received as a kind gift from Dr. Anne E. West from Duke University, Durham, NC [25]. The sequences for scr-sh and MeCP2 sh RNA are listed in the Table 1. Plasmid DNA was transformed into Stable3 competent bacterial cells as per the instructions from the manufacturer (Invitrogen, Grand Island, NY). Plamid DNA was extracted using a midi plasmid DNA extraction kit (Qiagen Valenica CA). Lentiviral plasmid DNA along with supporting plasmids were tranfected into HEK293 FT cells using lipofectamin LTX reagent following the instructions from the manufacturer (Invitrogen, Grand Island, NY). Lentiviral supernatant media samples produced from transfected cells were collected 48 h after transfection and centrifuged at 2000×g for 7 min and filtered through 0.4 µm filter. Lentivirus was concentrated by high-speed centrifugation at 25000 rpm at 4°C for 90 min in an ultra-centrifuge (Beckman, Brea, CA) in sterile PBS. The titer for concentrated lentivirus produced was determined by infecting the serially diluted virus along with transfection reagent polybrene to HEK293FT
