Embryoid body differentiation experiments for knock-in clones. (A) Results of EB differentiation. Incidence of hematopoietic differentiation of day-14 EBs derived from ES cell clones of wild-type (+/+), heterozygous for AML1-disruption (+/-), homozygous for the disruption (-/-), one knock-in allele for wild-type AML1 cDNA (-/AML1-KI), one knock-in allele for AML1-2 (-/AML1-2), and one knock-in allele for AML1-3 (-/AML1-3) genotypes in a representative experiment. Purple parts of the columns indicate the proportion of grown EBs with hematopoietic differentiation whereas pink parts represent those without hematopoietic elements. ES clones of the AML1-2 or AML1-3 knock-in allele developed hematopoietic cells in vitro as did control clones. (B) Appearance of representative day-14 EBs derived from the ES cell clones (top row). Morphology of the hematopoietic cells developed from ES cells of AML1-2 or AML1-3 knock-in clones showed no marked abnormalities of component cells examined with May-Grünwald-Giemsa staining (bottom row). Original magnifications: top row, × 20; bottom row, × 132. (C) Semiquantitative RT-PCR analysis of the total RNA from embryoid bodies was employed to identify recovered hematopoietic gene expression in the rescued clones. Messages for the G-CSF receptor (G-CSFR) and myeloperoxidase (MPO) genes were detected in the AML1-2– and AML1-3 clone–derived ES cells (lanes 5-8; results for 2 independent clones for each mutation are shown) as was the case for positive control clones (lanes 1, 2, and 4). On the other hand, a profound decrease in the expression for these genes was observed in the AML1-deficient (-/-) clone (lane 3). Expression of a housekeeping gene, HPRT, was assessed to provide a standard in parallel for the messenger RNA within the specimen. A dilution of 5^-0 of the RT specimen was used for PCR of G-CSFR and MPO and 5^-3 dilution for PCR of HPRT (see “Materials and methods”). bp indicates base pair.