Generation of Runx1-IRES-GFP knock-in mice. (A) To generate a targeting vector, the Runx1 cDNA at a unique SacII site was cloned in-frame into the corresponding SacII site of Runx1 exon 4, creating an artificial exon 4 that contains the entire 3′ coding region of the gene. This was followed by IRES-GFP and polyadenylation (pA) cassettes. There is approximately 10 kb of genomic DNA flanking Runx1 exon 4. The endogenous and targeted Runx1 alleles are also depicted. Filled rectangles indicate the 5′ and 3′ probes used for Southern analysis to detect homologous recombination. X denotes an XbaI site. (B) Southern blot analysis of genomic DNA isolated from the tails of offspring from a mating of 2 heterozygous Runx1-IRES-GFP knock-in mice. DNA was digested with XbaI. The wild-type and targeted alleles generate 14- and 9.8-kb bands, respectively, when detected with the 5′ probe.