A construct, which contains a lox66, exons 1 through 6 of human RUNX1 (hAML1), IRES, hyperactive variant of sleeping beauty (hsb5), polyadenylation sequence, FRT-flanked puro cassette, and lox71 site, was inserted into intron 5. Mice express a Etv6/RUNX1 fusion to replicate the TEL-AML1 translocation associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and hsb5. Flp-mediated recombination removed the puro cassette. Embryonic lethality indicates that no functional endogenous protein is produced despite the production of the endogenous transcript.