American Society of Hematology

Figure 6

$Figure 6. Expression of the Yall transgene but not of endogenous Mpl drops during megakaryocyte differentiation. (A) Comparison of transgenic (■) and endogenous () Mpl mRNA expression in megakaryocytes cultured for 0, 2, or 4 days with Tpo (error bars = SD). Bone marrow of mice containing both the Yall transgene and one wild-type Mpl allele (Yall;Mpl−/+) was lineage-depleted to enrich megakaryocytes and cultured in the presence of Tpo. Specific qPCR primers were used to distinguish mRNA of endogenous and transgene Mpl. Itga2b (CD41) mRNA was used as internal control, and data were normalized by arbitrarily setting one sample of the day 0 group to 100%. Results are based on bone marrow from 6 mice cultured separately. (B) Comparison of transgenic (■) and endogenous () Mpl in FACS-sorted megakaryocytes of low (R1) or high (R2) ploidy. Lineage-depleted bone marrow from Yall;Mpl−/+ mice was stained with FITC-labeled anti-CD41 antibodies and the DNA-binding dye Hoechst 33342. Only CD41+ cells are shown in the histogram. Megakaryocytes were sorted into 2 ploidy fractions—R1 (2n, 4n, 8n) and R2 (16n and higher)—based on their DNA content. The bar graph below indicates relative expression values measured as in panel A with the R1 value set to 100%.$

Expression of the Yall transgene but not of endogenous Mpl drops during megakaryocyte differentiation. (A) Comparison of transgenic (■) and endogenous () Mpl mRNA expression in megakaryocytes cultured for 0, 2, or 4 days with Tpo (error bars = SD). Bone marrow of mice containing both the Yall transgene and one wild-type Mpl allele (Yall;Mpl^−/+) was lineage-depleted to enrich megakaryocytes and cultured in the presence of Tpo. Specific qPCR primers were used to distinguish mRNA of endogenous and transgene Mpl. Itga2b (CD41) mRNA was used as internal control, and data were normalized by arbitrarily setting one sample of the day 0 group to 100%. Results are based on bone marrow from 6 mice cultured separately. (B) Comparison of transgenic (■) and endogenous () Mpl in FACS-sorted megakaryocytes of low (R1) or high (R2) ploidy. Lineage-depleted bone marrow from Yall;Mpl^−/+ mice was stained with FITC-labeled anti-CD41 antibodies and the DNA-binding dye Hoechst 33342. Only CD41⁺ cells are shown in the histogram. Megakaryocytes were sorted into 2 ploidy fractions—R1 (2n, 4n, 8n) and R2 (16n and higher)—based on their DNA content. The bar graph below indicates relative expression values measured as in panel A with the R1 value set to 100%.

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