Functional AML1-ETO fusion protein is detectable in the inducible founder lines.

(A) Western blot analysis of AML1-ETO expression in the bone marrow of transgenic mice. Transgenic mice from 5 unique founder lines positive for both MMTV-tTA and pUHD-AML1/ETO as well as one negative control mouse positive for MMTV-tTA but not pUHD-AML1/ETO were killed. Bone marrow was harvested from the femurs of these mice and resuspended in SDS-PAGE loading buffer. The marrow protein samples were electrophoresed in an 8% resolving SDS-PAGE gel (bis:acrylamide = 1:19). In addition to the protein from the bone marrow of the mice, 35 μg of nuclear extracts from wild-type Ba/F3 cells (a murine pro-B cell line) and Ba/F3 cells transfected with an AML1-ETO construct were electrophoresed as controls. The protein was then transferred to PVDF membrane and hybridized with an antibody against ETO. The blot was then developed by using a chemiluminescent substrate. The arrow points to the position of AML1-ETO. * marks a nonspecific band. (B) Northern blot analysis of UBP43 expression in inducible AML1-ETO mice. Transgenic mice that were positive for either pUHD-AML1/ETO (left side panels) or for MMTV-tTA and pUHD-AML1/ETO (right side panels) from founder line #7 were killed, and total RNAs were isolated from the bone marrow and peritoneal macrophages of these mice. Total RNA (10 μg) was loaded on each lane. The RNA was then transferred to a nylon membrane and hybridized sequentially with fragments of ETO cDNA and UBP43 cDNA. The ethidium bromide staining of the 28S ribosomal RNA is presented to show the loading of the RNA samples.