Fig. 1. Targeted disruption of the mouse Six1 gene. (A) Schematic representation of the wild-type allele, targeting vector (pPNT) and disrupted allele. The deleted sequence (starting in the 5' non coding region and extending to amino acid 178) codes for the N-terminal part of the Six1 protein, including the Six-domain and the Six-type homeodomain, both involved in specific DNA binding. The white and grey boxes represent the two exons of the Six1 gene with the coding region in grey; the blue box represents the ß-galactosidase reporter gene with the PGK-neomycin cassette downstream. The "NotI" site is a cloning site and thus is not present in the wild-type allele. (B) Phenotype of a newborn Six1^–/– mouse (left) and wild-type littermate (right). (C) Southern blot analysis of genomic DNA digested with NcoI and hybridized with a 5' external probe (left) and a 3' external probe (right). (D) Gel-mobility shift assays performed with total protein extracts from E12.5 Six1^+/+, Six1^+/– and Six1^–/– embryos, and with adult muscle nuclear extracts (amne) using a myogenin MEF3 probe. Different DNA/protein complexes can be identified. The amount of Six4 and Six5 DNA binding activity is not diminished in Six1^–/– extracts when compared to wild type, while no Six1 DNA binding activity is detected in Six1^–/– extracts. Six1 ab: added Six1 antibodies are able to displace specifically the Six1/MEF3 complex. ns: nonspecific protein/DNA interactions.