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 cells. The infected cells were observed under fluorescent microscopy for green fluorescence, since these viral vectors co-expresses green fluorescent protein (GFP) in their constructs. The titer was found to be 2×106 pfu/µl. In vitro validation of the MeCP2 sh-RNA lentivirus was performed by infecting the virus to primary pituitary PR1 cells. In vivo validation was performed by delivering the virus into the rat third ventricle and localizing the GFP labeled cells in the ARC.
