Fig. 2.
Fig. 2. Ectopic expression of. / Cbfb transgenes rescues definitive hematopoiesis by CBFβ-deficient ES cells. (A) Schematic diagram showing CBFβ isoforms generated by alternative splicing. Also shown is the experimentally truncated CBFβ(141) protein containing the heterodimerization domain for the CBFα proteins. Black rectangles indicate sequences within the heterodimerization domain. Sequences C-terminal to amino acid 165 in CBFβ(p22), CBFβ(p18), and CBFβ(p17.6) (stippled region) differ from those in CBFβ(p21.5). (B) Western blot analysis of CBFβ proteins produced by ectopic expression in Cbfb−/− ES cells. The genotype of the ES cell line and the protein produced from the ectopically expressedCbfb transgene is indicated above each lane. Protein lysates from 2 independently derived cell populations are shown. Arrow points to the endogenous CBFβ protein. Square and circle indicate positions of CBFβ(p18) and the truncated CBFβ(141) proteins, respectively. Molecular weight markers in kilodaltons are listed on the left. (C) Left panel: Definitive erythroid and mixed-lineage colonies generated in secondary cultures of EBs derived fromCbfb−/− ES cells ectopically expressing the CBFβ(p22) protein. Closed arrow indicates definitive erythroid colony; open arrow shows mixed-lineage colony. Right panel: Cytocentrifuge preparation of definitive mixed lineage colonies fromCbfb−/− ES cells ectopically expressing CBFβ(p22). Five mixed-lineage colonies were picked from secondary methylcellulose cultures, cytocentrifuged, and stained with Wright-Giemsa. Closed arrows identify myeloid cells, closed arrowheads show nucleated erythroid cells, and open arrowheads indicate enucleated red blood cells. (D) Globin expression analysis of secondary hematopoietic colonies. All colonies from one plate of a secondary culture of wild-type or Cbfb−/− EBs are compared to an individual definitive erythroid colony picked from a secondary culture of Cbfb−/− ES cells ectopically expressing CBFβ(141). Lanes 1, 4, and 7 show expression of βH1 (265 bp); lanes 2, 5, and 8 show βmajor (578 bp). HPRT expression (249 bp) is shown in lanes 3, 6, and 9 as a positive control. A 100-bp molecular weight ladder (M) flanks lanes 1 to 9. (E) Hematopoietic progenitor development in EBs from wild-type,Cbfb−/−, and Cbfb−/−ES cells expressing Cbfb transgenes. Colony assays were performed on EBs generated from 10 days of primary culture. The mean values of 2 or 3 independent plates are shown from one experiment. Similar results were obtained in 4 independent experiments. Differences in numbers of various colony types with expression of different transgenes were not statistically significant.

Ectopic expression of

Cbfb transgenes rescues definitive hematopoiesis by CBFβ-deficient ES cells. (A) Schematic diagram showing CBFβ isoforms generated by alternative splicing. Also shown is the experimentally truncated CBFβ(141) protein containing the heterodimerization domain for the CBFα proteins. Black rectangles indicate sequences within the heterodimerization domain. Sequences C-terminal to amino acid 165 in CBFβ(p22), CBFβ(p18), and CBFβ(p17.6) (stippled region) differ from those in CBFβ(p21.5). (B) Western blot analysis of CBFβ proteins produced by ectopic expression in Cbfb−/−ES cells. The genotype of the ES cell line and the protein produced from the ectopically expressedCbfb transgene is indicated above each lane. Protein lysates from 2 independently derived cell populations are shown. Arrow points to the endogenous CBFβ protein. Square and circle indicate positions of CBFβ(p18) and the truncated CBFβ(141) proteins, respectively. Molecular weight markers in kilodaltons are listed on the left. (C) Left panel: Definitive erythroid and mixed-lineage colonies generated in secondary cultures of EBs derived fromCbfb−/−ES cells ectopically expressing the CBFβ(p22) protein. Closed arrow indicates definitive erythroid colony; open arrow shows mixed-lineage colony. Right panel: Cytocentrifuge preparation of definitive mixed lineage colonies fromCbfb−/−ES cells ectopically expressing CBFβ(p22). Five mixed-lineage colonies were picked from secondary methylcellulose cultures, cytocentrifuged, and stained with Wright-Giemsa. Closed arrows identify myeloid cells, closed arrowheads show nucleated erythroid cells, and open arrowheads indicate enucleated red blood cells. (D) Globin expression analysis of secondary hematopoietic colonies. All colonies from one plate of a secondary culture of wild-type or Cbfb−/−EBs are compared to an individual definitive erythroid colony picked from a secondary culture of Cbfb−/−ES cells ectopically expressing CBFβ(141). Lanes 1, 4, and 7 show expression of βH1 (265 bp); lanes 2, 5, and 8 show βmajor (578 bp). HPRT expression (249 bp) is shown in lanes 3, 6, and 9 as a positive control. A 100-bp molecular weight ladder (M) flanks lanes 1 to 9. (E) Hematopoietic progenitor development in EBs from wild-type,Cbfb−/−, and Cbfb−/−ES cells expressing Cbfb transgenes. Colony assays were performed on EBs generated from 10 days of primary culture. The mean values of 2 or 3 independent plates are shown from one experiment. Similar results were obtained in 4 independent experiments. Differences in numbers of various colony types with expression of different transgenes were not statistically significant.

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