Generation of VenusYFP (Venus) knock-in ES cells. (A) Targeting strategy used to insert the Venus gene reporter under P2 proximal promoter of Runx1 locus. Exons are presented as boxes; P2-5′UTR is gray, coding regions are light. Black triangles are loxP sites; the 5′ external and internal genomic probes used for screening are indicated, as are the P2 proximal promoter of Runx1. SA-LacZ-stop, mouse Engrailed2 splice acceptor—LacZ—transcription stop cassette; neo, HSV-TK–neo^R–polyA cassette; pBlue, pBluescript II SK+; B, BamHI; X, XbaI; Xh, XhoI. (B) Southern blot analysis of the targeted allele. ES genomic DNA was digested by BamHI, blotted, and hybridized with the 5′-external genomic probe. The properly targeted allele generates a 2.4-kb DNA band. (C) Southern hybridization analysis of Runx1^fl/+ ES cells subjected to Cre recombinase–dependent excision of the floxed (loxP flanked) cassettes. Internal genomic probe hybridizes with 1.8-kb, 2.4-kb, and 8.8-kb XbaI genomic fragments derived from wild-type (+), Venus (V) knock-in, and floxed (fl) Runx1 alleles, respectively.