Introduction of an I-SceI restriction site proximal to the pseudoautosomal boundary (PAB). (A) Targeting strategy for the insertion of an I-SceI restriction site proximal to the PAB: wild-type Fxy allele (i), Fxy targeting vector (ii), and targeted Fxy allele (iii). The region of the gene surrounding exon two (black box) of the Fxy gene (toppanel) was replaced by a LacZ reporter gene (diagonal lines), which is in frame with the initiation codon (ATG), and a neoselection cassette (dark gray box) flanked by two LoxP sites (black triangles). An negative selection gene, Diphtheria toxin (dotted box), and an ampicillin resistance gene (light grey box), are located upstream of the 5′ homology region. The bottom panel shows the correctly targeted allele and the position of two probes used for Southern hybridization (probes A and B). Restriction enzyme sites indicated are A, AscI; B, BamHI; Bx, BstXI; En, EcoNI; I, I-SceI; N, NotI; and P,PacI. (B) Analysis of targeted clones by Southern hybridization. Genomic DNA from wild-type (WT) and targeted embryonic stem cells (Perry and Ashworth 1999) digested with restriction enzymes and hybridized to probe A (i), probe B (ii), or a probe derived from the neo gene (iii). Sizes estimated from molecular weight markers are indicated on the right.