Generation of mice overexpressing <i>Bmi1</i> in hematopoietic cells.

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