Murine thrombopoietin tpo

Human megakaryoblastic Meg-01 cells were cultured in RPMI 1640 medium at 37°C in 5% CO. Human 293T, hamster BHK-21, and mosquito C6/36 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) at 37°C in 5% CO₂, except for C6/36 cells, which were grown at 28°C. All media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 50 μg/mL gentamicin. Megakaryocyte differentiation from bone marrow cells of ProT Tg and C57BL/6 mice were maintained in DMEM supplemented with 10% FBS, 50 ng/mL murine thrombopoietin (TPO) (Peprotech, Cranbury, NJ), 10 ng/mL murine IL-3 (Peprotech), 2 mM L-glutamine, and 50 μg/mL gentamicin for 6 days.

BM cells were collected by flushing from femurs and tibias of a pool of male and female XCGD mice. Lin⁻ HSPCs isolated using the Lineage Cell Depletion Kit (Miltenyi) were cultured in Stem Span serum-free expansion medium (Stem Cell Technologies) supplemented with 1% l-glutamine, 1% penicillin/streptomycin, murine stem cell factor (SCF; 100 ng/mL), murine thrombopoietin (TPO; 50 ng/mL), human Flt3L (100 ng/mL), and human IL-3 (20 ng/mL; all from PeproTech) for 2 h. Cells were then cultured in the presence or absence (Mock) of a clinical preparation of MSP.Gp91.2T LV at an MOI of 200 for 16 ± 2 h.
Untransduced and transduced cells (1 × 10⁶/mouse) were injected intravenously in lethally (9 Gy split into two equal doses) irradiated 7- to 9-week-old XCGD male mice (XCGD GT mice, n = 20). Only male mice have been used in consideration of the fact that X-linked CGD usually affects only males. Mice were monitored for 52 weeks. Lin⁻ HSPCs isolated from XCGD mice and transduced or not with the MSP.Gp91.2T LV (0.5 × 10⁶/mouse) were injected intravenously in lethally (9 Gy split into two equal doses) irradiated 7- to 9-week-old WT CD45.1 male and female mice (XCGD GT WT mice, n = 12), and mice were monitored for 34 weeks.