Figure 1

Structural maps of the input plasmids and predicted targeted product (A): The target site plasmid, pACT-HYG, carries the hygromycin B phosphotransferase gene (hph), driven by the actin5C promoter from Drosophila (ACT) with transcription terminated by the SV40 polyadenylation signal (SV40). The hph fragment used as a probe for Southern analysis is indicated by a solid bar and all relevant restriction sites are shown (B, Bam HI; E, Eco RI; N, Nco I; S, Sph I). (B): The targeting vector plasmid, pH(NEO)YG, carries a copy of the hygromycin B phosphotransferase gene (hph), disrupted by insertion of a promoterless neomycin phosphotransferase coding sequence (neo). Following precise integration into the target site, transcription of the neo gene would be terminated by the SV40 polyadenylation signal (SV40). The neo fragment used as a probe for Southern analysis is indicated by a solid bar and all relevant restriction sites are shown (H, Hin dIII; E, Eco RI; S, Sph I; N, Nco I). (C): Structural map of the predicted targeted product with the neomycin resistance coding sequence (neo) inserted precisely into the hygromycin phosphotransferase target gene (hph) and expressed from the Drosophila actin5C promoter (ACT). The locations of the forward and reverse primers used in the PCR analysis are indicated by arrows and all relevant restriction sites are shown (E, Eco RI; S, Sph I; N, Nco I; B, Bam HI).