American Society of Hematology

Figure 1

$Figure 1. Retroviral introduction of BMI1 in human cord blood (CB) CD34+ cells. (A) Sorting strategies of nontransduced CB stem and progenitor fractions. (B) mRNA expression of BMI1 in human CB cells. Cells analyzed are: HSCs (CD34+CD38−), CMPs (CD34+CD38+IL3Rα+CD45RA−), MEPs (CD34+CD38+IL3Rα−CD45RA−), and GMs (CD34+CD38+IL3Rα+CD45RA+). (C) Schematic representation of the MiGR1 (control) and BMI1 retroviral vectors used in this study. (D) CB CD34+ cells were prestimulated for 48 hours in HPGM supplemented with KL, Flt3L, and TPO followed by 3 transduction rounds in the next 48 hours with MiGR1 or BMI1 retroviruses, and transduction efficiencies were determined on the basis of GFP expression by FACS. (E) Western blot analysis of total cell extracts from PG13 virus producer cells or CB CD34+ transduced cells. Membranes were probed with anti-BMI1 or anti-GFP antibodies. (F) Quantitative RT-PCR analysis of transduced cells for known downstream target genes of BMI1.$

Retroviral introduction of BMI1 in human cord blood (CB) CD34⁺ cells. (A) Sorting strategies of nontransduced CB stem and progenitor fractions. (B) mRNA expression of BMI1 in human CB cells. Cells analyzed are: HSCs (CD34⁺CD38⁻), CMPs (CD34⁺CD38⁺IL3Rα⁺CD45RA⁻), MEPs (CD34⁺CD38⁺IL3Rα⁻CD45RA⁻), and GMs (CD34⁺CD38⁺IL3Rα⁺CD45RA⁺). (C) Schematic representation of the MiGR1 (control) and BMI1 retroviral vectors used in this study. (D) CB CD34⁺ cells were prestimulated for 48 hours in HPGM supplemented with KL, Flt3L, and TPO followed by 3 transduction rounds in the next 48 hours with MiGR1 or BMI1 retroviruses, and transduction efficiencies were determined on the basis of GFP expression by FACS. (E) Western blot analysis of total cell extracts from PG13 virus producer cells or CB CD34⁺ transduced cells. Membranes were probed with anti-BMI1 or anti-GFP antibodies. (F) Quantitative RT-PCR analysis of transduced cells for known downstream target genes of BMI1.

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