Generation of Asxl1-MT KI mice. (A) Schematic depiction of the targeted Rosa locus, targeting vector, Asxl1-MT KI allele, and neostop deleted allele. The coding sequence 1–1,890 followed by stop codon was used in Asxl1 cDNA. DTA, diphtheria toxin A gene; EV, empty vector; GL, germline; SA, splicing acceptor. (B) Verification of correct homologous recombination. Using P1 and P2 primers in A, 3′ genomic PCR was performed. (C) Verification of correct homologous recombination of Asxl1-MT KI allele by a 5′ Southern blot using EcoRI-digested DNA and a 5′ external probe. (D) Confirmation of recombination after crossing with Vav-Cre transgenic mice using P3 and P4 primers (n = 4 mice per group). (E) Expected expression of FLAG-tagged Asxl1 mutant proteins in the BM and spleen cells. (F) GFP positivity of BM sample in Vav-Cre–negative Asxl1-MT^fl/fl (gray) and Vav-Cre–positive Asxl1-MT^fl/fl (red) mice. (G) DNA sequences of PCR products derived from Vav-Cre–positive Asxl1-MT^fl/fl BM cells. Both WT and KI sequences with corresponding amino acids are shown on the top. (H) Relative expression of N-terminal Asxl1 and C-terminal Asxl1 mRNA were measured by quantitative RT-PCR. Vav-Cre–negative Asxl1-MT^fl/fl (control) and Vav-Cre–positive Asxl1-MT^fl/fl BM cells were used (n = 3 per group). (I) HEK293T cells were transiently transfected with empty vector and FLAG-ASXL1 mutant (1900–1922del; E635RfsX15). Expression of ASXL1, FLAG-ASXL1 mutant, and Actin was analyzed by Western blot. Data are presented as mean ± SEM. **, P < 0.01 (Student’s t test). Experimental data were verified in at least two independent experiments. We used Vav-Cre–negative Asxl1-MT^fl/fl mice as littermate controls.