Fig. 1. Schematic view of the MTF-1 locus and targeting strategy. A transgene containing a mutant MTF-1 and the marker gene ‘white’ (w⁺) is circularized by FLP recombinase and linearized by the rare-cutter restriction enzyme I-SceI in germ cells. Alignment of the targeting DNA and resident MTF-1 locus by ‘ends-in’ recombination results in a duplication of MTF-1, with concomitant integration of the w⁺ gene into the MTF-1 locus. After identification of successful targeting events, the duplication is reduced to a single copy by means of homologous recombination, after inducing a double strand break in the genomic DNA with the other rare-cutter (I-CreI). The w⁺ marker gene is thereby lost. *, mutation of start colon.