Cloning of the AML1-TRPS1 that stimulates proliferation. (A) Spectral karyotyping (SKY) showed the balance translocation between chromosomes 8 and 21 (arrowheads). Representative SKY of chromosomes 8 and 21 is indicated as reverse DAPI (left) and SKY (right). (B) Fluorescence in situ hybridization using cosmid clones covering the whole AML1 gene showed that t(8;21)(q24;q22) involved the AML1 gene. Nearly half (55 [48.2%] of 114) of the cells had 3 signals of the AML1 gene. Images were acquired within an SD200 spectral imaging system (Applied Spectral Imaging, Migdal Haemek, Israel) attached to an Optiphot-2 epifluorescence microscope (Nikon, Tokyo, Japan) through a Splan Apo 100×/1.4 NA oil objective lens (Olympus, Tokyo, Japan) and analyzed with SkyView software (Applied Spectral Imaging). (C) The fusion partner gene with AML1 was cloned by long-distance 3′ RACE with 5′ primers on the AML1 exon 5 (AML1S5: 5′-cacagtggatgggccccgagaacctcg-3′) or exon 6 (AML1S6: 5′-tgcggcgcacagccatgagggtcagc-3′). The arrowhead indicates AML1a; the arrow shows the fusion gene. Lane 1, λ/HindIII DNA marker; lane 2, Human placental DNA; lane 3, AML cells (AML1S5); lane 4, AML cells (AML1S6). (D) N-terminal portion of AML1 retaining the Runt domain fused to the whole of TRPS1. An in-frame fusion gene consists of exon 6 of AML1 and TRPS1. In addition, sequencing analyses of subclones showed another transcript between exon 5 of AML1 and TRPS1, indicating skipping of exon 6 of AML1 and generating another in-frame fusion gene. The reciprocal TRPS1-AML1 transcript was not detected in the presenting case. (E) Structures of retrovirus constructs for control (MIG_mock) or AML1-TRPS1 (MIG_AT). AML1-TRPS1 was inserted into the indicated position of the plasmid MIG with internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (EGFP) gene. (F) Schematic depiction of the CFU-C assay. Fetal liver cells from embryonic day–14.5 (E14.5) mouse embryo were infected with the MIG vector. EGFP-positive cells were sorted and subjected to the CFU-C assay, supplemented with interleukin-3, stem cell factor, erythropoietin, and granulocyte colony-stimulating factor, as described previously.¹¹  (G) The number of all kinds of colony (total), granulocyte-macrophage (GM), and macrophage (M) are shown. Error bars indicate standard deviations for 3 independent experiments. Differences between AML1-TRPS1 (AT) and control (mock) transfectants were statistically significant (*P < .001, unpaired student t test) in all 3 categories.