Two-step culture experiments for in vitro hematopoietic rescue of knock-in ES cell clones. (A) Structure of the subdomain of the AML1 molecule and the C-terminal deletion mutants used for the knock-in experiments. Numbers represent positions of the residues. Runt indicates the runt domain; AD, trans-activation domain; ID, inhibitory domain; and VWRPY, VWRPY motif. (B) Targeted insertion of the cDNA into AML1-deficient ES cells, whose disrupted alleles are indicated by insertions of hygromycin-resistant (hygr) and neomycin-resistant (neo) cassettes at exon 4. Replacement-type vector for the targeted insertion and the schema of the resultant knock-in allele are shown at the bottom. pA indicates polyadenylation signal sequences; puro, puromycin-resistance cassette. (C) Capability for in vitro differentiation of the knock-in ES cell clones for each of the AML1 mutant cDNAs in a representative 2-step replating culture experiment (see “Materials and methods”). ES cells were cultured to form embryoid bodies. Hematopoietic progenitors of individual lineages that developed within embryoid bodies were then analyzed by the second-step culture on day 6 for primitive erythroid (Ery-P) and on day 10 for those of definitive origin, among them definitive erythroid (Ery-D), granulocyte-macrophage and macrophage (Myeloid), and mixed lineages including erythroid (E-Mix). Columns indicate numbers of progenitors per 105 disaggregated cells in culture.
