The lentivirus was surgically injected into the third ventricle of fetal alcohol exposed (AF) and control-fed rats (AD, PF) 60 days after birth. Rats were anaesthetized with sodium pentobarbital and placed on a stereotaxic frame. A small incision was made on the skin that overlies the skull. A small hole was drilled in the skull using stereotaxic coordinates (1.8 mm posterior from the bregma and 8 mm below the cortex). A Hamilton syringe carrying either scr-sh (control) or MeCP2-sh lentivirus was lowered in the hole through which 5 µl virus (10×106 pfu) was injected at the rate of 1 µl/min. After injection the syringe was kept in place for 5 min to avoid virus being sucked out during removal. The skin was closed with a wound clip. Rats were given two weeks time to recover and then used to collect the samples for gene transcript measurements by quantitative RT-PCR and gene methylation analysis by methylation specific PCR (MS-PCR).
