Fig. 4.
Fig. 4. Overexpression of. / Hoxa9 enhances self-renewal divisions of HSCs.(A) Evaluation of HSC numbers in bone marrow of Hoxa9-EGFPand EGFP chimeras using the CRU assay. Results are expressed as the mean ± SD of the CRU numbers in one femur ofHoxa9-EGFP (n = 2) and EGFP chimeras (n = 2) described in Table 1. (B-D) Southern blot analyses of proviral integration patterns in DNA isolated from bone marrow and thymus of the secondary recipients of (B) primary Hoxa9-EGFP (mouse Hoxa9-EGFP no. 1, Figure 1B); (C) primary EGFP mouse (mouse EGFP no. 1, Figure 1B) and (D) from a primary Hoxa9-EGFPrecipient receiving a lower dose on transduced cells (clonal analysis of this primary is not available). Secondary recipients were used to quantitate the CRU numbers and were all killed at 16 weeks posttransplantation. The DNA was digested with NcoI, which cuts once in the integrated provirus, generating a DNA fragment specific for each proviral integration site. The membranes were hybridized with an EGFP-specific probe to detect proviral fragments. Each secondary mouse is identified with a number. The number of bone marrow cells, as well as the estimated number of CRU cells received by each secondary recipient, is indicated above the blots. The percentages of EGFP expressing peripheral white blood cells in each of the secondary mice at 16 weeks after transplantation is indicated below each blot. The level of donor-derived (ie, Ly5.1+) peripheral blood leukocyte repopulation in these secondary mice shown were as follows: (B) 1.A1 (72%), 1.A2 (66%), 1.A3 (42%), 1.A4 (57%), 1.A5 (20%), 1.A6 (44%), 1.A7 (30%), 1.A8 (11%), 1.A9 (12%), 1.A10 (8%); (C) 1.E1 (11%), 1.E2 (14%), 1.E3 (10%), 1.E4 (15%), 1.E5 (5%), 1.E6 (0.5%); and (D) 2.A1 (55%), 2.A2 (25%), 2.A3 (47%), 2.A4 (12%), 2.A5 (15%), 2.A6 (11%), 2.A7 (5%), and 2.A8 (8%). The criteria used to define each stem cell clone are as follows: (i) same pattern of proviral integration in thymus (T cells) and bone marrow (myeloid and B cells); (ii) for clones containing more than one integrated provirus (eg, clone “a”), each band must be of the same intensity and always be transmitted together (eg, clone “a” in mouse 2.A3, 2.A1, 2.A8, and 2.A2 has 4 integrated proviruses; clone “b” also present in bone marrow of mouse 2.A2 is different because it does not always transmit with clone “a”; see thymus in mouse 2.A1 or mouse 2.A8 for examples). B indicates bone marrow; T, thymus; PBL, peripheral blood leukocytes.

Overexpression of

Hoxa9 enhances self-renewal divisions of HSCs.(A) Evaluation of HSC numbers in bone marrow of Hoxa9-EGFPand EGFP chimeras using the CRU assay. Results are expressed as the mean ± SD of the CRU numbers in one femur ofHoxa9-EGFP (n = 2) and EGFP chimeras (n = 2) described in Table 1. (B-D) Southern blot analyses of proviral integration patterns in DNA isolated from bone marrow and thymus of the secondary recipients of (B) primary Hoxa9-EGFP (mouse Hoxa9-EGFP no. 1, Figure 1B); (C) primary EGFP mouse (mouse EGFP no. 1, Figure 1B) and (D) from a primary Hoxa9-EGFPrecipient receiving a lower dose on transduced cells (clonal analysis of this primary is not available). Secondary recipients were used to quantitate the CRU numbers and were all killed at 16 weeks posttransplantation. The DNA was digested with NcoI, which cuts once in the integrated provirus, generating a DNA fragment specific for each proviral integration site. The membranes were hybridized with an EGFP-specific probe to detect proviral fragments. Each secondary mouse is identified with a number. The number of bone marrow cells, as well as the estimated number of CRU cells received by each secondary recipient, is indicated above the blots. The percentages of EGFP expressing peripheral white blood cells in each of the secondary mice at 16 weeks after transplantation is indicated below each blot. The level of donor-derived (ie, Ly5.1+) peripheral blood leukocyte repopulation in these secondary mice shown were as follows: (B) 1.A1 (72%), 1.A2 (66%), 1.A3 (42%), 1.A4 (57%), 1.A5 (20%), 1.A6 (44%), 1.A7 (30%), 1.A8 (11%), 1.A9 (12%), 1.A10 (8%); (C) 1.E1 (11%), 1.E2 (14%), 1.E3 (10%), 1.E4 (15%), 1.E5 (5%), 1.E6 (0.5%); and (D) 2.A1 (55%), 2.A2 (25%), 2.A3 (47%), 2.A4 (12%), 2.A5 (15%), 2.A6 (11%), 2.A7 (5%), and 2.A8 (8%). The criteria used to define each stem cell clone are as follows: (i) same pattern of proviral integration in thymus (T cells) and bone marrow (myeloid and B cells); (ii) for clones containing more than one integrated provirus (eg, clone “a”), each band must be of the same intensity and always be transmitted together (eg, clone “a” in mouse 2.A3, 2.A1, 2.A8, and 2.A2 has 4 integrated proviruses; clone “b” also present in bone marrow of mouse 2.A2 is different because it does not always transmit with clone “a”; see thymus in mouse 2.A1 or mouse 2.A8 for examples). B indicates bone marrow; T, thymus; PBL, peripheral blood leukocytes.

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