Efficient and Rapid Induction of a Chronic Myelogenous Leukemia-Like Myeloproliferative Disease in Mice Receiving P210 bcr/abl-Transduced Bone Marrow

Abstract

Expression of the 210-kD bcr/abl fusion oncoprotein can cause a chronic myelogenous leukemia (CML)-like disease in mice receiving bone marrow cells transduced by bcr/abl-encoding retroviruses. However, previous methods failed to yield this disease at a frequency sufficient enough to allow for its use in the study of CML pathogenesis. To overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210 bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The myeloproliferative disease recapitulates many of the hallmarks of human CML and is characterized by high white blood cell counts and extensive extramedullary hematopoiesis in the spleen, liver, bone marrow, and lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green fluorescent protein shows that the vast majority of bcr/abl-expressing cells are myeloid. Analysis of the proviral integration pattern shows that, in some mice, the myeloproliferative disease is clonal. In multiple mice, the CML-like disease has been transplantable, inducing a similar myeloproliferative syndrome within 1 month of transfer to sublethally irradiated syngeneic recipients. The disease in many of these mice has progressed to the development of acute lymphoma/leukemia resembling blast crisis. These results demonstrate that murine CML recapitulates important features of human CML. As such, it should be an excellent model for addressing specific issues relating to the pathogenesis and treatment of this disease.

Stat5 is indispensable for the maintenance of bcr/abl ‐positive leukaemia

“…Stat5 is required for bcr/abl p210 -induced myeloid leukaemia maintenance in vivo Transplantation of bcr/abl p210þ BM cells is a reliable method to develop a fatal rapidly progressing myeloproliferative illness in mice which is commonly determined 'CML-like' disease (Pear et al, 1998;Van Etten, 2001). We retrovirally transduced BM cells from 5-fluorouracil (FU) pretreated Stat5 fl/fl Mx1Cre and Stat5 fl/fl mice with bcr/abl p210 IRES GFP and injected them i.v.…”

TLX1/HOX11 transcription factor inhibits differentiation and promotes a non‐haemopoietic phenotype in murine bone marrow cells

“…In order to gain further insight into the nature of this widespread interference with haemopoietic cell differentiation, we examined the effects of constitutive TLX1 expression on adult murine BM cells, both in vitro and in vivo, following retroviral transduction. To facilitate the tracking of transduced cells and their descendants, a bicistronic vector enabling long terminal repeat (LTR)-driven expression of TLX1 and GFP (MigR1/TLX1) was constructed using the MigR1 retroviral backbone (Pear et al, 1998). The parental vector containing GFP alone (MigR1) was used as the negative control.…”

A potential role for the Src‐like adapter protein SLAP‐2 in signaling by the colony stimulating factor‐1 receptor

“…Thus full-length SLAP-2-FLAG was ectopically expressed in bone marrow macrophages in order to establish if SLAP-2 modulates signaling by the CSF-1 receptor. Briefly, the retroviral vector MIGR1 [26] was used to transduce SLAP-2-FLAG into mouse bone marrow progenitor cells and macrophages subsequently grown out from the cultures. Because green fluorescent protein (GFP) is coexpressed in cells transduced by MIGR1-derived retroviruses [26], macrophages that had been transduced with the SLAP-2-FLAG or control empty retrovirus were purified by FACS-based cell sorting prior to biochemical analysis.…”

“…Briefly, the retroviral vector MIGR1 [26] was used to transduce SLAP-2-FLAG into mouse bone marrow progenitor cells and macrophages subsequently grown out from the cultures. Because green fluorescent protein (GFP) is coexpressed in cells transduced by MIGR1-derived retroviruses [26], macrophages that had been transduced with the SLAP-2-FLAG or control empty retrovirus were purified by FACS-based cell sorting prior to biochemical analysis. Despite our ability to co-immunoprecipitate the CSF-1 receptor with SLAP-2-FLAG when both proteins were transiently coexpressed in HEK293T cells (see Fig.…”

“…The PCR product generated was subcloned in-frame into a pcDNA3-based plasmid to create pcDNA3-SLAP-2-FLAG and sequenced to ensure the absence of mutations. The retroviral expression vector MIGR1-SLAP-2-FLAG was created by excising the cDNA from pcDNA3-SLAP-2-FLAG with EcoRI and XbaI, blunting with Klenow, and subcloning the fragment into the HpaI site in MIGR1 [26]. Retroviral vectors expressing SH2 and SH3 mutants of SLAP-2-FLAG (i.e.…”