Targeting strategy to introduce knock-in alleles expressing chimeric AML1 genes with heterologous C-terminal portion. (A) Structure of Runx family molecules and chimeric AML1 with AML2- or AML3-derived C-terminus (AML1-2 and AML1-3, respectively). Numerals indicate the positions of amino acid residues of the molecules as shown in Figure 1B. □ indicates parts derived from AML1, ▦ from AML2, and from AML3. (B) Integrity of the mutant construct was confirmed by Western blot analysis of transiently transfected COS-7 cells. Arrows indicate protein bands detected by rabbit antisera against N-terminal peptide of AML1. (C) Biochemical activities of AML1 or the mutant molecules were examined by means of reporter assay experiments. Each of pRc effector constructs and pM-CSF-R-luc were cotransfected into HeLa cells with CBFβ and phRL-TK control vectors. The height of the columns indicates the increase in relative luciferase activity on the M-CSF receptor gene promoter (see “Materials and methods”). Bars indicate standard deviations of triplicate experiments. Both of the chimera proteins retained trans-activation activity although the activity of AML1-3 for this promoter construct was somewhat weaker than that of AML1b or AML1-2. Three independent experiments were performed with basically the same results. Representative results are shown. (D) Targeting strategy to introduce knock-in allele for AML1-2 or AML1-3 into ES cell of AML1-deficient genotype. Both alleles of exon 4, which corresponds to the middle of the Runt domain, had been disrupted by insertion of KO(hygr) (the hygromycin-resistance cassette) and KO(neo) (the neomycin-resistance cassette). Replacement-type (knock-in) vectors were designed to introduce KI(puro) (the knock-in allele). □ indicates non-coding regions of the exons; ▪, coding exons; , runt domain; ▧, poly-adenylation signal sequences; ▤, DNA fragments for the probes. (E) Clones subjected to homologous recombination are detectable by Southern blot analysis.