Figure 4
PCR analysis of the targeted site in the HT-1 cell line The forward primer was sited at the 3' end of the Drosophila actin5C promoter and the reverse primer was located at the 5' end of the neomycin resistance coding sequence. Amplification took place over 30 cycles (95°C, 30 seconds; 50°C, 30 seconds; 72°C, 1 minute) in a total volume of 50 μl containing 1 × PCR buffer, 2 mM MgCl2, 200 μM each dNTP, 0.2 μM each primer and either 50 ng high molecular weight DNA or 1 ng plasmid DNA template. The upper panel shows an agarose gel analysis of 20 μl reaction products from AH-4 cellular DNA template (Lane 1); targeting vector plasmid (pH(NEO)YG) template (Lane 2) and HT-1 cellular DNA template (Lane 3). Lane M carries molecular size markers (MBI 100 bp ladder). The lower panel shows a structural map of the amplified region with the neomycin resistance coding sequence (neo) inserted into the hygromycin resistance gene (shown in red) downstream of the Drosophila actin5C promoter (ACT) with transcription terminated by the SV40 polyadenylation signal (SV40). The PCR primers (arrowed) can only amplify a product of 850 bp in the event of targeted integration of neo into the hph gene.