Figure 1.

Construction of the Mll-AF4 KI allele and characterization of targeted ES cells. (A) A diagrammatic description of the KI allele. The top map represents the Mll wild-type allele, indicating the 8-kb fragment detected by N-P1.6 probe. The bottom map represents the Mll-exon7-AF4–targeting allele, indicating the position of the Mll-AF4 breakpoint, PGK-Neo cassette, and a 12-kb fragment detected by N-P1.6 probe in Southern blotting. The Mll-AF4 breakpoint and junction sequence are also shown. (B) Southern blotting analysis of wild-type and targeted ES cells. The 8-kb wild-type (WT) band and 12-kb KI band are indicated. (C) Detection of Mll-AF4 fusion gene by PCR. PCR was performed with gDNA from ES cells with KI allele (lane 1), wild-type ES cells (lane 2), and a representative Mll-AF4 mouse (lane 3). The 5′ primer of Mll exon 6 and 3′ primer of human AF4 amplified a 930-bp Mll-AF4 fragment in lanes 1 and 3. (D) Expression of Mll-AF4 fusion protein in targeted ES cells detected by Western blotting. The 240-kDa Mll-AF4 fusion protein was detected in targeted ES cells. A cell line transfected with MLL-AF4 DNA was used as positive control. (E) Mll-AF4, Mll-AF9, and wild-type mouse at the age of 5 weeks. Mll-AF4 mice have a shortened face and large ears. Mll-AF9 mice have a more pointed face and large ears.