C57BL/6J mice were superovulated at 3 to 4 weeks of age, and embryos were collected. High-titer lentivirus (>107 pg p24 antigen/ml) in a volume of 10 to 100 pl was injected into the perivitelline space of single-cell embryos using a pulled glass pipette, as described in Lois and colleagues (2002) and implanted into pseudopregnant CD1 females. Of 141 injected embryos, we obtained 63 pups, 39 derived from shLmo3.8 injections and 24 derived from shScr injections. To confirm genomic insertion of the lentivirus, we isolated DNA from tail clips using standard methods. PCR amplification was performed with primers against the pLL3.7 vector and green fluorescent protein (GFP) encoded by the lentiviral vector. Primer sequences are listed in the Supplemental Table S1. Of the 63 pups analyzed, 13 (6 females, 7 males) of 39 (33%) shLmo3.8-injected animals and 9 (5 females, 4 males) of 24 (37.5%) shScr-injected animals were positive for virus by genotyping. Twelve shLmo3.8 and 8 shScr animals were used for behavioral testing.
