The mice/D171N with integration near Evi1 site were monoclonal. (A) Southern blot analysis of mice/D171N. DNA samples were digested with EcoRI, which cut the retrovirus only once within the multicloning site. Probes used were DNA fragments of the GFP coding sequence. Mouse IDs are shown at the top of the panel. (B) The mice/D171N + Evi1 were polyclonal. DNA samples were digested with EcoRI. Proviruses were probed with a GFP probe. (C) Real-time PCR for Evi1 in BM derived from morbid mice/D171N or mice/S291fs or mice/WT or mice/mock. In addition to 6 samples from mice/D171N harboring integration near Evi1 (mouse IDs 4, 6, 7, 12, 14, and 15), 4 samples derived from mice/D171N without integration near Evi1 display high expression levels of Evi1 (mouse IDs 13, 19, 20, and 22). RNA from normal BM cells served as a control (RNA level = 1). (D) Western blot of lysates from spleen cells of mice/D171N, mice/WT, and normal mice and PLAT-E as controls. Samples from mice/D171N confirmed high expression of Evi1 by RT-PCR showed expression of the protein (mouse IDs 6, 15, 20, and 22), but the other mice without high expression of Evi1 by RT-PCR did not express the protein (mouse IDs 11, 17, and 105).