Generation of knock-in ES cells expressing

Cbfb-GFP. (A) Targeting scheme used to generateCbfb^+/GFP ES cells. The construct contains exon 5 (e5) of Cbfb fused in frame to GFP. The positive selection marker is SV40-Hygro; the negative selection marker is PGK-TK. Exon 4 (e4) is in the genomic sequence 5′ to the targeting vector. Correctly targeted ES cell clones express Cbfb-GFP under the control of the endogenous Cbfb promoter. (B,C) Southern blot analysis of DNA isolated from 3 independently targeted ES cell lines. DNA was digested with either NcoI (B) or XbaI (C). The external probe (0.2C) hybridized to a 3′ genomic fragment and detected a 15.7-kb NcoI band from the wild-type allele and a 6.3-kb NcoI band from the targeted allele (B). The internal probe (Hygro) hybridized to thehygromycin gene and detected a single 7.4-kb band in the targeted allele (C). (D) Western blot analysis using a monoclonal antibody against Cbfβ (1-141) demonstrated expression of endogenous Cbfβ (22 kDa) or the Cbfβ fusion proteins in 3 ES cell lines. TC-1 is the wild-type ES cell line (lane 1); Cbfb-MYH11KI no. 55 is an ES cell clone that expresses Cbfβ-SMMHC (lane 2); Cbfb-GFP no. 52 is one of the correctly targeted ES cell clones expressing Cbfβ-GFP (lane 3).