Generation of a conditional mouse model with the human NPM1 mutation A. (A) Strategy for targeted insertion of a human NPM1 mutation A cDNA into the murine ROSA26 genomic DNA locus. The figure describes the structure of the mouse ROSA26 locus (top), the targeting vector (upper middle), the targeted allele (lower middle), and Cre-mediated excised allele (bottom). The targeting vector was a 16-kb plasmid containing a transgenic cassette expressing the NPM1 mutant cDNA (NPM1; *position of the TCTG duplication), a combined loxP flanked STOP-Neomycin selection cassette, an inducible CAG promoter, and a negative DTA selection marker. NEO is excised through Cre-mediated recombination between the 2 loxP sites (triangles). Positions of the 5′ and 3′ probes used to confirm the homologous recombination and relevant restriction sites for Southern blot analysis are indicated. (B) Southern blotting analysis confirmed the presence of homologous recombination in Npm1-TCTG/WT mice (top) and ES cells (bottom). (C) PCR genotyping strategy for Npm-TCTG/WT mice to distinguish the wild-type allele (304 bp) from the knocked-in recombined allele (472 bp).