Generation of mice overexpressing Bmi1 in hematopoietic cells.

(A) Strategy for making a knock-in allele for Bmi1 downstream of the Rosa26 promoter. A loxP-flanked neo^r-stop cassette followed by Flag-tagged Bmi1, an frt-flanked IRES-eGFP cassette, and a bovine polyadenylation sequence was knocked-in the Rosa26 locus. (B) Quantitative RT-PCR analysis of Bmi1 in BM LSK cells from Tie2-Cre and Tie2-Cre;R26Stop^FLBmi1 mice. mRNA levels were normalized to Hprt1 expression. Expression levels relative to that in Tie2-Cre LSK cells are shown as the mean ± S.D. (n = 3). (C) Western blotting analysis of Bmi1 in c-Kit⁺ BM cells from Tie2-Cre and Tie2-Cre;R26Stop^FLBmi1 mice. α-tubulin was used as the loading control. (D) Hematopoietic analysis of 10-week-old Tie2-Cre and Tie2-Cre;R26Stop^FLBmi1 mice. Absolute numbers of BM cells, CD34^-LSK cells, and LSK cells in bilateral femurs and tibiae are presented as the mean ± S.D. (upper panels, Tie2-Cre; n = 7, Tie2-Cre;R26Stop^FLBmi1; n = 8). PB analysis of 10-week-old Tie2-Cre and Tie2-Cre;R26Stop^FLBmi1 mice. White blood cell (WBC) counts and lineage contribution of myeloid, B, and T cells are shown as the mean ± S.D. (lower panels, Tie2-Cre; n = 7, Tie2-Cre;R26Stop^FLBmi1; n = 8). ** p<0.01.