Fig. 1.
Fig. 1. A 7-day culture system that eliminates acute lethal myeloproliferation induced by the transplantation of bone marrow cells engineered to overexpress BCR-ABL. / (A) Schematic representation of the different retroviruses used in these studies. Selector genes were the enhanced green fluorescent protein (EGFP), the red fluorescent protein (RFP), or the neomycin-resistance gene. B indicates BamHI; Bg,BglII; RI, EcoRI; K, KpnI; N,NheI; X, XbaI. RFP in the selector gene shown for the restriction mapping of the NUP98-HOXA9 andHOXA9 retroviruses. (B) Description of the 7-day culture protocol used to eliminate the acute myeloproliferative syndrome induced by transplanting BM cells engineered to overexpressBCR-ABL. (C) Survival curve of recipients of 2 × 105 BCR-ABL–transduced BM cells grown (solid line) or not (dashed line) for 7 days in vitro with hemopoietic growth factors. (D) Cytological analysis of bone marrow (BM), liver (Li), spleen (SPL), and peripheral blood (PB) specimens showing the acute and lethal myeloproliferative disease developing in recipients of BCR-ABL–transduced cells transplanted immediately following coculture on viral producers. The spleen is infiltrated by myeloid precursors (EGFP+) that are barely detectable in the PB. Original magnification × 40; stain, Wright Giemsa. (E) Southern blot analyses demonstrating the integrated provirus (top blots) in the DNA isolated from the BM (B) or spleen (S) of recipients of BCR-ABL–transduced cells (nos. 1-5). DNA was digested with XbaI (panels E and G). The bands smaller than 9.7 kb probably represent several clones that harbor rearranged proviruses. Panels F and H depict Northern blot analyses to demonstrate expression level of BCR-ABL in the various samples as detailed in panels E and G. T indicates thymus. Exposure times are 12 hours for panel F and 8 days for panel H. 18S RNA probe is exposed for the same time for all lanes. The “minus” lane indicates 10 μg RNA isolated from spleen-derived cells from an unmanipulated syngenic mouse. Panel G shows Southern blot analysis of genomic DNA isolated from recipients that received 2 × 105(day “0”) BCR-ABL–transduced BM cells grown for 7 days in vitro prior to transplantation. Note the large variation in exposure time from 12 hours for mice 1 to 5 to 3 days for mice 5A to 7A. The mouse identification numbers in this figure correspond to those shown in Table 1. Single copy is from the endogenous actingene: exposure times for membranes exposed to actin are identical. The “−” lane indicates 10 μg DNA isolated from the spleen of a syngenic control mouse, and the “+” sign is a positive control for hybridization consisting of 20 pg KpnI-digested retroviral plasmid no. 652 (“Materials and methods”) generating a 2.7-kb fragment.

A 7-day culture system that eliminates acute lethal myeloproliferation induced by the transplantation of bone marrow cells engineered to overexpress BCR-ABL.

(A) Schematic representation of the different retroviruses used in these studies. Selector genes were the enhanced green fluorescent protein (EGFP), the red fluorescent protein (RFP), or the neomycin-resistance gene. B indicates BamHI; Bg,BglII; RI, EcoRI; K, KpnI; N,NheI; X, XbaI. RFP in the selector gene shown for the restriction mapping of the NUP98-HOXA9 andHOXA9 retroviruses. (B) Description of the 7-day culture protocol used to eliminate the acute myeloproliferative syndrome induced by transplanting BM cells engineered to overexpressBCR-ABL. (C) Survival curve of recipients of 2 × 105BCR-ABL–transduced BM cells grown (solid line) or not (dashed line) for 7 days in vitro with hemopoietic growth factors. (D) Cytological analysis of bone marrow (BM), liver (Li), spleen (SPL), and peripheral blood (PB) specimens showing the acute and lethal myeloproliferative disease developing in recipients of BCR-ABL–transduced cells transplanted immediately following coculture on viral producers. The spleen is infiltrated by myeloid precursors (EGFP+) that are barely detectable in the PB. Original magnification × 40; stain, Wright Giemsa. (E) Southern blot analyses demonstrating the integrated provirus (top blots) in the DNA isolated from the BM (B) or spleen (S) of recipients of BCR-ABL–transduced cells (nos. 1-5). DNA was digested with XbaI (panels E and G). The bands smaller than 9.7 kb probably represent several clones that harbor rearranged proviruses. Panels F and H depict Northern blot analyses to demonstrate expression level of BCR-ABL in the various samples as detailed in panels E and G. T indicates thymus. Exposure times are 12 hours for panel F and 8 days for panel H. 18S RNA probe is exposed for the same time for all lanes. The “minus” lane indicates 10 μg RNA isolated from spleen-derived cells from an unmanipulated syngenic mouse. Panel G shows Southern blot analysis of genomic DNA isolated from recipients that received 2 × 105(day “0”) BCR-ABL–transduced BM cells grown for 7 days in vitro prior to transplantation. Note the large variation in exposure time from 12 hours for mice 1 to 5 to 3 days for mice 5A to 7A. The mouse identification numbers in this figure correspond to those shown in Table 1. Single copy is from the endogenous actingene: exposure times for membranes exposed to actin are identical. The “−” lane indicates 10 μg DNA isolated from the spleen of a syngenic control mouse, and the “+” sign is a positive control for hybridization consisting of 20 pg KpnI-digested retroviral plasmid no. 652 (“Materials and methods”) generating a 2.7-kb fragment.

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