Figure 2. Recapitulation of MYC/IgH and BCR/ABL1 translocations.

A, Site1 vector (red triangle on chromosome 14) was targeted to the IGHA2 locus of Nalm-6 cells expressing tamoxifen-regulated Cre. Site2 vector (red triangle on chromosome 8) was subsequently targeted to the MYC locus (red circle). The IgH enhancer is indicated by a green circle. B, Flow cytometric profiles of the indicated stages. Red circles indicate the sorted cell populations. C, Confirmation of translocation by PCR. PCR products of expected sizes were observed for cells after translocation (post) but not for cells before Cre expression (pre). H₂O lanes show the no-template controls. Triangles above the transgene schemes represent primer positions (IgA2-B41-F1 and MYC-B23-R1 for green, and MYC-B41-F1 and IgA2-B23-R1 for red), respectively. D, FISH analysis demonstrating MYC/IgH translocation for cells after recombination by Flp, using BAC probes. Green probe hybridizes to a chromosome 14 region near Site1-integrated IgH locus on the centromeric side. Red probe hybridizes to a chromosome 8 region near Site2-integrated MYC locus on the telomeric side. These probes co-localize only after Cre-mediated translocation and appear as a yellow signal (right panel, box), magnification of which is shown in the inset. E, Site1 and Site2 vectors containing loxP sites (red rectangle) were targeted into the first intron of the BCR gene and the ABL1 gene, respectively. Vertical bars and boxes connected with v-shaped lines indicate exon–intron structure of genes. The derivative chromosome 22 [der(22)] recapitulates the Philadelphia (Ph) chromosome. F, SKY analysis of cells with artificial BCR/ABL1 translocation. The derivative chromosome 9 [der(9)] in the green box contains the material (pink) translocated from chromosome 22. The amount of chromosome 9 material that was translocated to chromosome 22 was too small to be resolved by this SKY analysis (Ph chromosome, pink box). The previously reported translocation between chromosomes 5 and 12 in Nalm-6 cells [23] could be also detected. G, Western blot confirming expression of the BCR-ABL1 fusion protein (190 kD) after clean translocation (post) with constitutive expression of ABL1 and GAPDH proteins.