Generation of the Etv6-RUNX1 transposase knockin allele (Etv6^+/RUNX1). (A) The endogenous Etv6 locus was targeted to introduce a SA, exons 1 to 6 of human RUNX1, an IRES followed by a hyperactive variant of the SB transposase (HSB5), and an FRT-flanked PGK-PuroΔTK drug selection marker between exons 5 and 6. The entire cassette was flanked by Lox66 and Lox71 sites for potential Cre-mediated inactivation of the Etv6-RUNX1 fusion gene. The PuroΔTK drug marker was removed by breeding mice to an Flpe deleter strain before tumor watch experiments. Probe A (5′) and probe B (3′) are shown. S, StuI. (B) Southern blot analysis on StuI-digested tail DNA confirmed germ line transmission and homologous recombination of the 5′ and 3′ targeting vector arms. (C) RT-PCR showing expression of Etv6-RUNX1 fusion transcripts in bone marrow from Etv6^+/RUNX1 mice, using primers located as indicated by the arrows on the schematic of the Etv6 locus. Bottom panel shows a sequence trace showing splicing of the Etv6 and RUNX1 transcripts to produce an in-frame fusion transcript. (D) qPCR of Etv6 and Etv6-RUNX1 fusion transcripts (using primers as mentioned herein). RNA was extracted from bone marrow, spleen, and thymus. Relative Etv6 gene expression in wild-type (blue) and Etv6^+/RUNX1 tissues (red), and relative Etv6-RUNX1 fusion transcript expression in Etv6^+/RUNX1 tissues (green) is shown. The data were collected from the analysis of 5 littermate mice of each genotype (± SD). **P < .005, *P < .05.