Figure 1
Figure 1. Phenotypic and functional characterization of HSCs in Gata3fl/fl Vav-Cretg/+ mice. (A) Gata3 mRNA expression in 1-week-old WT LSKFLT3−CD150+CD48− HSCs, LSKFLT3hiIL7R− lymphoid-primed multipotent progenitors, and Lin−CD25−KIT+FLT3+ early thymic progenitors. Data are expressed as mean (SEM) normalized Robust Multi-Array Averages (RMA) expression (“Gene expression analysis”). n = 3 experiments. (B) BM cellularity in 1-week-old Gata3fl/flVav-Cre+/+ and Gata3fl/flVav-Cretg/+ mice. Data are mean (SD); n = 3 or 4 mice/genotype. ns indicates not significant. (C) Fluorescence-activated cell sorter profiles from representative mouse showing gating strategy for LSKCD150+CD48− cells in 1-week-old mice. Numbers indicate percentage of total BM cells within the indicated gate. n = 3 or 4 mice/genotype. (D) Bar graphs represent mean (SD) frequency of LSKCD150+CD48− cells. n = 3 or 4 mice/genotype. ns indicates not significant. (E) Bar graphs show mean (SEM) Gata3 mRNA expression levels (relative to Hprt) in LSKCD150+CD48− HSCs isolated from 1- to 2-week-old Gata3fl/fl Vav-Cre+/+ or Gata3fl/fl Vav-Cretg/+ mice. (F-I) A total of 0.5 to 2 million BM cells from 1- to 2-week-old Gata3fl/fl Vav-Cre+/+ or Gata3fl/fl Vav-Cretg/+ mice (CD45.2) were transplanted into lethally irradiated (900 cGy) WT (CD45.1) recipients together with a similar dose of competitor WT (CD45.1) adult BM cells. (F) Representative fluorescence-activated cell sorter analysis of peripheral blood 4 months after transplantation. Numbers indicate percentage of indicated gate within reconstituted T (CD4+/CD8+), B (CD19+), and myeloid (Gr1+Mac1+) cells. n = 3 to 6 mice/group. (G-H) Mean (SEM) percentage of test cell (CD45.2+) reconstitution within the T, B, and myeloid blood cell lineages, respectively, 4 to 6 (G) or 11 (H) months after transplantation. (G) n = 6 to 9 mice/group. (H) n = 2 or 3 mice/group. (I) Six months after primary transplantation, one-half femur equivalent from each primary recipient was transplanted into one secondary WT (CD45.1) recipient. Secondary recipients were analyzed 8 months later. Data show mean (SEM) percentage of test cell reconstitution within T, B, and myeloid reconstituted cells, respectively. N = 3 to 6 mice/group. 1° indicates primary reconstitution; and 2°, secondary reconstitution. *P < .05. ***P < .001. ns indicates not significant.

Phenotypic and functional characterization of HSCs in Gata3fl/fl Vav-Cretg/+ mice. (A) Gata3 mRNA expression in 1-week-old WT LSKFLT3−CD150+CD48− HSCs, LSKFLT3hiIL7R− lymphoid-primed multipotent progenitors, and Lin−CD25−KIT+FLT3+ early thymic progenitors. Data are expressed as mean (SEM) normalized Robust Multi-Array Averages (RMA) expression (“Gene expression analysis”). n = 3 experiments. (B) BM cellularity in 1-week-old Gata3fl/flVav-Cre+/+ and Gata3fl/flVav-Cretg/+ mice. Data are mean (SD); n = 3 or 4 mice/genotype. ns indicates not significant. (C) Fluorescence-activated cell sorter profiles from representative mouse showing gating strategy for LSKCD150+CD48− cells in 1-week-old mice. Numbers indicate percentage of total BM cells within the indicated gate. n = 3 or 4 mice/genotype. (D) Bar graphs represent mean (SD) frequency of LSKCD150+CD48− cells. n = 3 or 4 mice/genotype. ns indicates not significant. (E) Bar graphs show mean (SEM) Gata3 mRNA expression levels (relative to Hprt) in LSKCD150+CD48− HSCs isolated from 1- to 2-week-old Gata3fl/fl Vav-Cre+/+ or Gata3fl/fl Vav-Cretg/+ mice. (F-I) A total of 0.5 to 2 million BM cells from 1- to 2-week-old Gata3fl/fl Vav-Cre+/+ or Gata3fl/fl Vav-Cretg/+ mice (CD45.2) were transplanted into lethally irradiated (900 cGy) WT (CD45.1) recipients together with a similar dose of competitor WT (CD45.1) adult BM cells. (F) Representative fluorescence-activated cell sorter analysis of peripheral blood 4 months after transplantation. Numbers indicate percentage of indicated gate within reconstituted T (CD4+/CD8+), B (CD19+), and myeloid (Gr1+Mac1+) cells. n = 3 to 6 mice/group. (G-H) Mean (SEM) percentage of test cell (CD45.2+) reconstitution within the T, B, and myeloid blood cell lineages, respectively, 4 to 6 (G) or 11 (H) months after transplantation. (G) n = 6 to 9 mice/group. (H) n = 2 or 3 mice/group. (I) Six months after primary transplantation, one-half femur equivalent from each primary recipient was transplanted into one secondary WT (CD45.1) recipient. Secondary recipients were analyzed 8 months later. Data show mean (SEM) percentage of test cell reconstitution within T, B, and myeloid reconstituted cells, respectively. N = 3 to 6 mice/group. 1° indicates primary reconstitution; and 2°, secondary reconstitution. *P < .05. ***P < .001. ns indicates not significant.

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