To begin the investigation of whether the mammalian Lmo genes are involved in behavioral responses to ethanol, we targeted Lmo3 in the mouse using RNAi. Lmo3 is 1 of 4 mammalian homologs of dLmo, and is expressed at high levels in brain regions important for behavioral responses to ethanol, particularly in the forebrain (Bulchand et al., 2003; Hinks et al., 1997). We designed 3 independent shRNAs specific for Lmo3 (Fig. 2A). To test their ability to reduce Lmo3 expression, lentiviral plasmids encoding each Lmo3 shRNA (shLmo3) were transfected into mouse Neuro-2a cells in parallel to the control shScr plasmid, which encodes a scrambled shRNA sequence that does not target any mouse gene (Lasek et al., 2007). Two days after transfection, RNA was isolated and subjected to first-strand cDNA synthesis and real-time qPCR. We found that Lmo3 levels were significantly reduced by 30 to 43% in cells transfected with each of the shLmo3 plasmids compared with the shScr control (Fig. 2B). Transfection of shLmo3 plasmids had no detectable effect on the expression of the closely related Lmo family members Lmo4 (Fig.
