BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation
Figure 6
Lineage Analyses and clonality of Tumor Cells.
(A) T-cell receptor β gene rearrangement. Genomic DNA isolated from bone marrow of three primary mice diagnosed with leukemia/lymphoma (#25, 32, and 33) and expressing HA-tagged BCL2A1a were subjected to a Southern blot analysis using a Cβ2 probe able to recognize the Cβ1 and Cβ2 region of the T-cell receptor β gene. Germline genomic DNA should produce bands of 8.9 and 2.9 Kb when digested with HindIII, and a band of 11.1 Kb when digested with SacI. NIH3T3 cells (N) were used as a control. Ladder size is given in base pairs. (B, C) Immunoglobulin gene rearrangements. PCRs to assess V to DJ rearrangements were performed with primers identifying the degenerated heavy variable region (VH558 and VH7183) as forward primers and J3 (B) or J4 (C) primers recognizing J3 or J4 genes. PCRs were performed on genomic DNA isolated from the bone marrow of control mice (MOCK #3 and vector #7), three primary mice diagnosed with leukemia/lymphoma (#25, 32, and 33), and one secondary mouse also diagnosed with leukemia (#24-12 expressing HA-tagged BCL2A1a). Genomic DNA obtained from BaF3 cells (B) and regular bone marrow from C57BL/6 mouse (M) were used as controls as well as a no-template control with water (W). Ladder size is given in base pairs, and are designated by a white dot. White arrows indicate the J gene involved in the rearrangement. The complete panel of PCRs for V to DJ and V to J rearrangements as well as germline configurations are shown in Supplemental Figure S4. (D) Southern blot performed with a GFP probe on genomic DNA isolated from whole bone marrow cells of primary mice. Genomic DNA obtained from regular bone marrow from C57BL/6 mouse (M) and from K562/D33 (K) harboring one copy of pRRL.PPT.SF.IRES.GFPpre/HA were respectively used as negative and positive controls. Control mice MOCK #3 and vector #7 were also used as controls. #25, 32, 33, 28, and 26 were primary mice diagnosed with leukemia/lymphoma. Ladder size is given in base pairs. (E) Southern blot performed with GFP probe on genomic DNA isolated from whole bone marrow (BM), lymph node (LN), or peripheral blood (PB) cells of secondary mice. Genomic DNA obtained from BaF3 cells (B) and from K562/AL (K) harboring two copy of GFP vector were respectively used as negative and positive controls. Plasmid pRRL.PPT.SF.IRES.GFPpre/HA-BCL2A1a was also used as a positive control. Mice vector #7 and primary BCL2A1a #25 were used as controls. Secondary BCL2A1a #29-16, 35-18, 35-19, and vector #20-9 mice belong to the first set of secondary transplant. Secondary BCL2A1a mouse #23-11 belongs to the second set of secondary transplant. All secondary mice were diagnosed with lymphoma/leukemia. Ladder size is given in base pairs.