Targeting of the D11S12 locus. (A; Top) The genomic 5.5-kbEcoRI fragment includes a 5.0-kb BamHI-targeted region. (Middle) Scheme of the pVT-3 targeting vector linearized with EcoRI and NotI. Yeast sequences,CEN6, ARSH4, and TRP1, and the mammalianZeo R gene are shown. The vector can replace the D11S12 region by double crossover. (Bottom) Structure of the targeted locus. As a result of the targeting replacement, the size of the EcoRI fragment becomes 8.5 kb. (B) Southern blots of EcoRI-digested DNA from the parental DT40 11-3 line (lane1) and five drug-resistant clones (lanes2–6). DNA was probed with a 5′ external probe (a 5′ external 0.5-kb fragment of the D11S12 locus), showing a 5.5-kb parental fragment and the expected 8.5-kb targeted fragment in three of the five clones (lanes 4–6). The same filter was probed with the Zeo probe (a 1.3-kbNaeI-Zeo R gene-containing fragment), showing an 8.5-kb Zeo R-containing fragment in the same three clones (lanes 10–12). (E) EcoRI; (Ba)BamHI; (B) BclI.
