Fig. 1. Generation of a Foxl2^lacZ allele by homologous recombination. (A) Genomic structure of Foxl2 locus showing the single coding exon (red box) containing the WH/forkhead domain (green box). The targeting vector contains a lacZ cassette (blue box) in frame with the first ATG and a loxP-flanked neomycin-resistance (pACN) cassette replacing sequences encoding amino acids 62-375 and part of the 3' untranslated region. A diphteria toxin (DTA) cassette was included for negative selection. A, Asp718; B, BsaBI; N1, NheI; N2, NcoI; S, SpeI; X, XhoI. (B) Southern blot analysis of genomic tail DNA probed with a 5'- and 3'-outside probe, respectively. Genomic DNA was digested with Asp718/SpeI. The 5' probe detects the expected 12.2 kb WT band and the 7.8 kb mutant band, whereas the 3' probe detects the 12.2 kb WT band and a 6.6 kb fragment after removal of the selection cassette. (C) Protein was extracted from P1 ovaries of WT and mutant mice and analysed by western blot analysis using the anti-Foxl2 antibody directed against the N-terminus of Foxl2. In WT extracts a doublet band was detected at around 50 kDa. This doublet band disappears in Foxl2 mutant ovary extracts. Instead the Foxl2LacZ fusion protein is detected at 125 kDa.