Murine Retroviral Bone Marrow Transplantation Models for the Study of Human Myeloproliferative Disorders

L. Cristina Gavrilescu1, Richard A. Van Etten1

1 Molecular Oncology Research Institute, Tufts Medical Center, Boston, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.10
DOI:  10.1002/0471141755.ph1410s43
Online Posting Date:  December, 2008
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Abstract

Human myeloproliferative diseases are common hematologic disorders characterized by clonal overproduction of maturing myeloid or erythroid cells, often caused by expression of a mutant, dysregulated tyrosine kinase (TK). These diseases can be accurately modeled in laboratory mice by the retroviral transfer of a mutant TK gene into murine hematopoietic stem and progenitor cells, followed by transplantation of these cells into irradiated recipient mice. This yields a model system for analyzing the molecular pathophysiology of these conditions and provides a platform for testing therapies, particularly molecularly targeted new chemical entities (NCEs). The Basic Protocol in this unit describes the preparation of mouse bone marrow cells to express the relevant human oncogene before transplanting them into irradiated recipient mice. An alternate protocol describes a similar technique that allows specific induction of lymphoproliferative disease by some TKs. Support protocols for generating and titering retroviral stocks are also included. Curr. Protoc. Pharmacol. 43:14.10.1‐14.10.28. © 2008 by John Wiley & Sons, Inc.

Keywords: BCR‐ABL; chronic myeloid leukemia; JAK2; lymphoma; mouse model; polycythemia vera

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Bone Marrow Transplantation for Induction of Chronic Myeloproliferative Syndromes in Mice
  • Alternate Protocol 1: Direct Retroviral Infection and Bone Marrow Transplantation to Selectively Induce B‐Lymphoid Leukemia with BCR‐ABL
  • Support Protocol 1: Production of Retrovirus Stock by Transient Transfection of HEK 293T Cells
  • Support Protocol 2: Checking Transfection Efficiency and Virus Production
  • Support Protocol 3: Titering Virus Stocks by Flow Cytometry, Drug Selection, or Southern Blotting
  • Support Protocol 4: Preparing WEHI‐3B Cell‐Conditioned Medium (WEHI‐CM)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Bone Marrow Transplantation for Induction of Chronic Myeloproliferative Syndromes in Mice

  Materials
  • Donor and recipient mice (see Critical Parameters)
    • Donors: 6‐ to 10‐week‐old male BALB/c mice (from Jackson Laboratory or Taconic Farms)
    • Recipients: 6‐ to 8–week‐old female BALB/c mice (from Jackson Laboratory or Taconic Farms)
  • 5 to 10 mg/ml 5‐fluorouracil (5‐FU) stock solution (Sigma, cat. no. 6627), prepared fresh in phosphate‐buffered saline (PBS; 5‐FU is poorly soluble, and agitating the solution at 37°C helps to dissolve it)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Cellgro, cat. no. 21‐040‐CV)
  • Ice
  • Bone marrow flush medium (see recipe)
  • 70% ethanol
  • Erythrocyte lysis solution (see recipe or can be purchased from Sigma, cat. no. R7757)
  • Trypan blue solution
  • Ficoll‐Paque (Stem Cell Technologies, cat. no. 7907)
  • Bone marrow prestimulation medium (see recipe)
  • Cytokines (see Critical Parameters)
    • Recombinant murine IL‐3 (1000× = 6 µg/ml; PeproTech, cat. no. 213‐13)
    • Recombinant murine IL‐6 (500× stock = 5 µg/ml; PeproTech, cat. no. 216‐16)
    • Recombinant murine SCF (1000× = 50 µg/ml; PeproTech, cat. no. 250‐0)
  • Virus spinfection solution (see recipe)
  • Oncogene‐expressing retroviral stocks (see protocol 3)
  • 800 µg/ml polybrene (hexadimethrine bromide; Sigma, H‐9268) stock in PBS store at −20°C
  • 1 M HEPES, pH 7.4 (Invitrogen, cat. no. 15630‐080)
  • Hanks' Balanced Salt Solution without Ca2+/Mg2+ (HBSS, sterile; Cellgro Cat, cat. no. 21‐023‐CV)
  • Acidified water (pH 1.3 to 2.0, or between 2 to 10 ml conc. 38% HCl per liter of water)
  • Autoclaved chow
  • Tissue culture dishes (6‐cm and 10‐cm)
  • Bucket
  • 10‐ml sterile syringes, luer‐lock
  • Single‐use 27‐G, 1/2‐in. needles
  • Dissection tools (from Roboz) including:
    • Curved dissection scissors
    • Toothed forceps
    • Bone rongeurs
  • Styrofoam dissecting board
  • 21‐G needles
  • Gloves
  • 50‐ml conical tissue culture tubes
  • Sterile tissue culture pipets (2‐ml, 5‐ml, 10‐ml, and 25‐ml)
  • Sorvall RT‐6000 centrifuge or equivalent, with a swinging‐bucket plate holder and HB1000B rotor, or equivalent
  • 6‐well tissue culture plates
  • Parafilm
  • Radiation source
  • Wide‐mouth pipet
  • U‐100 insulin syringes, 27‐G needle, 1.0‐ml volume
  • Microisolator cages
  • Rodent hematology analyzer (e.g., HemaVet, CDC Coporation)
  • Additional reagents and equipment for parenteral injections (Donovan and Brown, ), rodent euthanasia (Donovan and Brown, ), performing a viable cell count using a hemacytometer and trypan blue staining (Phelan, ), and blood collection from rodents (Donovan and Brown, )

Alternate Protocol 1: Direct Retroviral Infection and Bone Marrow Transplantation to Selectively Induce B‐Lymphoid Leukemia with BCR‐ABL

  Materials
  • 293T cells (ATCC)
  • 293T cell medium (see recipe)
  • 0.05% Trypsin/EDTA (CellGro, cat. no. 25‐051‐CI)
  • 25 mM chloroquine (1000× stock; Sigma, cat. no. C6628) in PBS (store up to 6 months at −20°C)
  • Retroviral oncogene‐expressing vector [e.g., pMIG RI (Pear et al., ); see page 14.10.12]
  • TE (Tris‐EDTA) buffer, pH 8.0 ( appendix 2A)
  • Packaging plasmid [e.g., pMCV‐ecopac (Finer et al., ; available from the authors) or pCL (Naviaux et al., ; available from Dr. Inder Verma, Salk Institute)]
  • 2 M CaCl 2 in dH 2O, sterile filtered (store up to 6 months at room temperature)
  • 2× HBS, pH 7.05 (see recipe)
  • 6‐cm tissue culture plates
  • 15‐ml conical tube
  • P1000 pipettor
  • Suction pump
  • 10‐ml sterile syringes
  • Single‐use 18‐G needles
  • 0.22‐µm syringe filter
  • 50‐ml conical tubes
  • 2‐ and 4‐ml screw‐top cryovials

Support Protocol 1: Production of Retrovirus Stock by Transient Transfection of HEK 293T Cells

  Materials
  • 293T cells transfected with pMFG‐lacZ plasmid DNA (available from the authors; CsCl‐purified, in TE buffer) together with the pMCV–Ecopac packaging construct (see protocol 3)
  • Phosphate‐buffered saline calcium‐ and magnesium‐free (CMF‐PBS)
  • 25% glutaraldehyde (50× stock; store up to 2 years in the dark at −20°C)
  • 1 M MgCl 2 (500× stock in dH 2O; Sigma, cat. no. M8266; store at room temperature)
  • 0.7M K 3Fe(CN) 6 (20× stock potassium ferricyanide dissolved in dH 2O; Sigma, cat. no. P8131; store up to 6 months in the dark at room temperature)
  • 0.7M K 4Fe(CN) 6.3H 2O (20× stock potassium ferrocyanide dissolved in dH 2O; Sigma, cat. no. P9387; store up to 6 months in the dark at room temperature)
  • 50 mg/ml X‐Gal (50× stock in N,N–DMF; Sigma, cat. no. B9146; store up to 1 year in the dark at −20°C)
  • Additional reagents and equipment for harvesting the viral supernatant ( protocol 3)

Support Protocol 2: Checking Transfection Efficiency and Virus Production

  Materials
  • NIH 3T3 cells (ATCC)
  • 3T3 cell medium (see recipe)
  • Retroviral stocks (e.g., MSCV‐IRES/GFP or MSCV‐IRES/Neo vectors; see protocol 3), frozen
  • 800 µg/ ml polybrene (100× stock; Sigma, cat. no. H9268); store at −20°C
  • Sterile phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; CellGro, cat. no. 21‐040‐CV)
  • 0.05% trypsin/EDTA (CellGro, cat. no. 25‐051‐CI)
  • FACS buffer (see recipe)
  • 1 mg/ml neomycin in H 2O (G418; Sigma, cat. no. G1279); store at −20°C
  • Crystal Violet solution [1% (w/v) crystal violet in 20% methanol]
  • 2× DNA lysis buffer (see recipe)
  • Proteinase K (solid; Roche, cat. no. 1245500)
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol (25:24:1)
  • 24:1 (v/v) chloroform:isoamyl alcohol
  • 3M sodium acetate, pH 5.2
  • Isopropanol
  • 70% ethanol
  • TE buffer, pH 8.0 ( appendix 2A)
  • Restriction enzyme (e.g., Xba I)
  • DNase‐free RNase A (100 µg/ml stock)
  • DNA loading buffer (Voytas, )
  • 0.8% agarose/TBE slab gel (Voytas, )
  • Radioactive probe
  • 6‐cm tissue culture dishes
  • 37°C, 10% CO 2 incubator
  • 1.5‐ml microcentrifuge tubes
  • Fluorescence‐activated cell analyzer (e.g., FACSCalibur; Becton Dickinson)
  • Benchtop microcentrifuge
  • 37°C water bath
  • Pasteur pipet with end heat‐sealed and melted into a small hook
  • Sorvall RT‐6000 centrifuge
  • Nylon membrane
  • X‐ray film or phosphor imager screen
  • Additional reagents and equipment for DNA extraction ( appendix 3C), determining DNA concentration by spectrophotometry (Gallagher and Desjardins, ), agarose gel electrophoresis (Voytas, ), Southern blotting (Southern, ), electroblotting (Li et al., ), and quantitation of radiolabeled DNA (Voytas and Ke, )

Support Protocol 3: Titering Virus Stocks by Flow Cytometry, Drug Selection, or Southern Blotting

  Materials
  • WEHI‐3B cells (ATCC) in culture
  • Growth medium (DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin)
  • 175‐cm2 flasks
  • 50‐ml sterile plastic tubes
  • 0.45‐µm filter system
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Figures

  •   FigureFigure 14.10.1 Dissection and removal of femur and tibia. (A) Dissection equipment setup. (B) Location of patellar tendon. (C) Position of tibia.
  •   FigureFigure 14.10.2 Bone marrow flushing procedure. (A) Equipment setup. (B) Flushing of marrow out of bone with a syringe of bone marrow flushing solution.
  •   FigureFigure 14.10.3 Histopathology of BCR‐ABL‐induced myeloproliferative disease in mice. (A) Peripheral blood smear (800×, Wright‐Giemsa stain), demonstrating increased maturing myeloid cells with metamyelocytes, myelocytes, and occasional blast forms. (B) Liver (200×, Hematoxylin‐eosin stain), demonstrating periportal infiltration with maturing myeloid and erythroid cells. (C) Spleen (200×, Hematoxylin‐eosin stain), demonstrating disruption of follicular architecture with maturing myeloid cells. (D) Lung (75×, Hematoxylin‐eosin stain), demonstrating alveolar hemorrhage and infiltration with myeloid cells.
  •   FigureFigure 14.10.4 Flow cytometric dot‐plot profiles of peripheral blood leukocytes from a mouse with CML‐like disease induced by a retroviral vector co‐expressing BCR‐ABL and GFP. GFP fluorescence is on the x axis, while the y axis depicts intensity of PE‐conjugated antibodies against different hematopoietic cell surface antigens. This mouse had leukocytosis with a peripheral blood leukocyte count of 150,000/µl, the majority of which were myeloid cells expressing Mac‐1 (CD11b) and Gr‐1. The fraction of cells in the upper/outer quadrant is displayed. Note the small population of GFP‐negative myeloid cells, which probably represents malignant cells that have down‐regulated or lost expression of GFP (see Li et al., ).
  •   FigureFigure 14.10.5 Top: Kaplan‐Meier survival curve for bone marrow recipient mice with CML‐like MPD induced by BCR‐ABL (red curve) and the aggressive MPD induced by BCR‐FGFR1 (green curve). Mutation of the Grb2 binding site at BCR Tyr177 greatly attenuates the latter disease (brown curve). Bottom: Scatter plot of peripheral blood leukocyte count (y axis, logarithmic scale) as a function of time after transplantation (x axis) for the recipients depicted in the top panel. Reproduced with permission from Roumiantsev et al. (). Abbreviations: PB WBC, peripheral blood white blood cells; BMT, bone marrow transplant.
  •   FigureFigure 14.10.6 Titering GFP‐expressing retrovirus stocks by flow cytometry. NIH 3T3 cells either untransduced or transduced with the indicated dilution of retrovirus stock were assessed 48 hour post‐transduction for expression of GFP ( x axis).

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Literature Cited

Literature Cited
   Barnes, D.J., Schultheis, B., Adedeji, S., and Melo, J.V. 2005. Dose‐dependent effects of Bcr‐Abl in cell line models of different stages of chronic myeloid leukemia. Oncogene 24:6432‐6440.
   Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N., Swanton, S., Vassiliou, G.S., Bench, A.J., Boyd, E.M., Curtin, N., Scott, M.A., Erber, W.N., Green, A.R., and Cancer Genome Project 2005. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365:1054‐1061.
   Bhattacharya, D., Rossi, D.J., Bryder, D., and Weissman, I.L. 2006. Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning. J. Exp. Med. 203:73‐85.
   Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 68:2.9.1‐2.9.20.
   Cherry, S.R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., and Jaenisch, R. 2000. Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol. Cell. Biol. 20:7419‐7426.
   Cools, J., DeAngelo, D.J., Gotlib, J., Stover, E.H., Lagare, R.D., Cottes, J., Kutok, J., Clark, J., Galinsky, I., Griffin, J.D., Cross, N.C., Tefferi, A., Malone, J., Alam, R., Schrier, S.L., Schmid, J., Rose, M., Vandenberghe, P., Verhoef, G., Boogaerts, M., Wlodarska, I., Kantarjian, H., Marynen, P., Coutre, S.E., Stone, R., Gilliland, D.G. 2003. A tyrosine kinase created by fusion of the PDGFA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N. Engl. J. Med. 348:1201‐1214.
   Daley, G.Q., Van Etten, R.A., and Baltimore, D. 1990. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 247:824‐830.
   Dameshek, W. 1951. Some speculations on the myeloproliferative disorders. Blood 6:372‐375.
   Gallagher, S.R. and Desjardins, P.R. 2006. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Mol. Biol. 76:A.3D.1‐A.3D.21.
   Demiroglu, A., Steer, E.J., Heath, C., Taylor, K., Bentley, M., Allen, S.L., Koduru, P., Brody, J.P., Hawson, G., Rodwell, R., Doody, M.L., Carnicero, F., Reiter, A., Goldman, J.M., Melo, J.V., and Cross, N.C. 2001. The t(8;22) in chronic myeloid leukemia fuses BCR to FGFR1: Transforming activity and specific inhibition of FGFR1 fusion proteins. Blood 98:3778‐3783.
   Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Donovan, J. and Brown, P. 2006c. Blood collection. Curr. Protoc. Immunol. 73:1.7.1‐1.7.9.
   DuBridge, R.B., Tang, P., Hsia, H.C., Leong, P.M., Miller, J.H., and Calos, M.P. 1987. Analysis of mutation in human cells by using an Epstein‐Barr virus shuttle system. Mol. Cell. Biol. 7:379‐387.
   Fialkow, P.J., Jacobson, R.J., and Papayannopoulou, T. 1977. Chronic myelocytic leukemia: Clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage. Am. J. Med. 63:125‐130.
   Finer, M.H., Dull, T.J., Qin, L., Farson, D., and Roberts, M. 1994. kat: A high‐efficiency retroviral transduction system for primary human T lymphocytes. Blood 83:43‐50.
   Fioretos, T., Panagopoulos, I., Lassen, C., Swedin, A., Billstrom, R., Isaksson, M., Strombeck, B., Olofsson, T., Mitelman, F., and Johansson, B. 2001. Fusion of the BCR and the fibroblast growth factor receptor‐1 (FGFR1) genes as a result of t(8;22)(p11;q11) in a myeloproliferative disorder: The first fusion gene involving BCR but not ABL. Gene. Chromosome Canc. 32:302‐310.
   Fletcher, F.A., Moore, K.A., Ashkenazi, M., DeVries, P., Overbeek, P.A., Williams, D.E., and Belmont, J.W. 1991. Leukemia inhibitory factor improves survival of retroviral vector‐infected hematopoietic stem cells in vitro, allowing efficient long‐term expression of vector‐encoded human adenosine deaminase in vivo. J. Exp. Med. 174:837‐845.
   Furitsu, T., Tsujimura, T., Tono, T., Ikeda, H., Kitayama, H., Koshimizu, U., Sugahara, H., Butterfield, J.H., Ashman, L.K., and Kanayama, Y. 1993. Identification of mutations in the coding sequence of the proto‐oncogene c‐kit in a human mast cell leukemia cell line causing ligand‐independent activation of c‐kit product. J. Clin. Invest. 92:1736‐1744.
   Golub, T.R., Barker, G.F., Lovett, M., and Gilliland, D.G. 1994. Fusion of the PDGF receptor b to a novel ets‐like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 77:307‐316.
   Griffin, J.H., Leung, J., Bruner, R.J., Caligiuri, M.A., and Briesewitz, R. 2003. Discovery of a fusion kinase in EOL‐1 cells and idiopathic hypereosinophilic syndrome. Proc. Natl. Acad. Sci. U.S.A. 100:7830‐7835.
   Hanenberg, H., Xiao, X.L., Dilloo, D., Hashino, K., Kato, I., and Williams, D.A. 1996. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat. Med. 2:876‐882.
   Hardy, R.R., Carmack, C.E., Shinton, S.A., Kemp, J.D., and Hayakawa, K. 1991. Resolution and characterization of pro‐B and pre‐pro‐B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213‐1225.
   Heisterkamp, N., Jenster, G., Kioussis, D., Pattengale, P.K., and Groffen, J. 1991. Human bcr‐abl gene has a lethal effect on embryogenesis. Transgenic Res. 1:45‐53.
   Hu, Y., Liu, Y., Pelletier, S., Buchdunger, E., Warmuth, M., Fabbro, D., Hallek, M., Van Etten, R.A., and Li, S. 2004. Requirement of Src kinases Lyn, Hck and Fgr for BCR‐ABL1‐induced B‐lymphoblastic leukemia but not chronic myeloid leukemia. Nat. Genet. 36:453‐461.
   Hu, Y., Swerdlow, S., Duffy, T.M., Weinmann, R., Lee, F.Y., and Li, S. 2006. Targeting multiple kinase pathways in leukemic progenitors and stem cells is essential for improved treatment of Ph+ leukemia in mice. Proc. Natl. Acad. Sci. U.S.A. 103:16870‐16875.
   Huettner, C.S., Zhang, P., Van Etten, R.A., and Tenen, D.G. 2000. Reversibility of acute B‐cell leukaemia induced by BCR‐ABL1. Nat. Genet. 24:57‐60.
   Inhorn, R.C., Aster, J.C., Roach, S.A., Slapak, C.A., Soiffer, R. Tantravahi, R., and Stone, R.M. 1995. A syndrome of lymphoblastic lymphoma, eosinophilia, and myeloid hyperplasia/malignancy associated with t(8;13)(p11;q11): Description of a distinctive clinicopathological entity. Blood 85:1881‐1887.
   Jaiswal, S., Traver, D., Miyamoto, T., Akashi, K., Lagasse, E., and Weissman, I.L. 2003. Expression of BCR/ABL and BCL2 in myeloid progenitors leads to myeloid leukemias. Proc. Natl. Acad. Sci. U.S.A. 100:10002‐10007.
   James, C., Ugo, V., Le Couedic, J.P., Staerk, J., Delhommeau, F., Lacout, C., Garcon, L., Raslova, H., Berger, R., Bennaceur‐Griscelli, A., Villeval, J.L., Constantinescu, S.N., Casadevall, N., and Vainchenker, W. 2005. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434:1144‐1148.
   Jiang, X., Stuible, M., Chalandon, Y., Li, A., Chan, W.Y., Eisterer, W., Krystal, G., Eaves, A., and Eaves, C. 2003. Evidence for a positive role of SHIP in BCR‐ABL‐mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia. Blood 102:2976‐2984.
   Jones, A.V. and Cross, N.C. 2004. Oncogenic derivatives of platelet‐derived growth factor receptors. Cell. Mol. Life Sci. 61:2912‐2923.
   Kotani, H., Newton, P.B. 3rd, Zhang, S., Chiang, Y.L., Otto, E., Weaver, L., Blaese, R.M., Anderson, W.F., and McGarrity, G.J. 1994. Improved methods of retroviral vector transduction and production for gene therapy. Hum. Gene Ther. 5:19‐28.
   Kralovics, R., Passamonti, F., Buser, A.S., Teo, S.S., Tiedt, R., Passweg, J.R., Tichelli, A., Cazzola, M., and Skoda, R.C. 2005. A gain‐of‐function mutation of JAK2 in myeloproliferative disorders. N. Engl. J. Med. 352:1779‐1790.
   Krause, D.S. and Van Etten, R.A. 2004. Adoptive immunotherapy of BCR‐ABL‐induced chronic myeloid leukemia‐like myeloproliferative disease in a murine model. Blood 104:4236‐4244.
   Krause, D.S., and Van Etten, R.A. 2005. Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med. 353:172‐187.
   Lacout, C., Pisani, D.F., Tulliez, M., Gachelin, F.M., Vainchenker, W., and Villeval, J.L. 2006. JAK2V617F expression in murine hematopoietic cells leads to MPD mimicking human PV with secondary myelofibrosis. Blood 108:1652‐1660.
   Lee, J.C., Hapel, A.J., and Ihle, J.N. 1982. Constitutive production of a unique lymphokine (IL 3) by the WEHI‐3 cell line. J. Immunol. 128:2393‐2398.
   Levine, R.L., Wadleigh, M., Cools, J., Ebert, B.L., Wernig, G., Huntly, B.J., Boggon, T.J., Wlodarska, I., Clark, J.J., Moore, S., Adelsperger, J., Koo, S., Lee, J.C., Gabriel, S., Mercher, T., D'Andrea, A., Fröhling, S., Döhner, K., Marynen, P., Vandenberghe, P., Mesa, R.A., Tefferi, A., Griffin, J.D., Eck, M.J., Sellers, W.R., Meyerson, M., Golub, T.R., Lee, S.J., and Gilliland, D.G. 2005. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7:387‐397.
   Li, S., Ilaria, R.L., Million, R.P., Daley, G.Q., and Van Etten, R.A. 1999. The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia‐like syndrome in mice but have different lymphoid leukemogenic activity. J. Exp. Med. 189:1399‐1412.
   Li, S., Gillessen, S., Tomasson, M.H., Dranoff, G., Gilliland, D.G., and Van Etten, R.A. (2001). Interleukin‐3 and granulocyte‐macrophage colony‐stimulating factor are not required for induction of chronic myeloid leukemia‐like myeloproliferative disease in mice by BCR/ABL. Blood 97:1442‐1450.
   Macdonald, D., Aguiar, R.C., Mason, P.J., Goldman, J.M., and Cross, N.C. 1995. A new myeloproliferative disorder associated with chromosomal translocations involving 8p11: A review. Leukemia 9:1628‐1630.
   Macdonald, D., Reiter, A., and Cross, N.C.P. 2002. The 8p11 myeloproliferative syndrome: A distinct clinical entity caused by constitutive activation of FGFR1. Acta. Haematol. 107:101‐107.
   McLaughlin, J., Chianese, E., and Witte, O.N. 1987. In vitro transformation of immature hematopoietic cells by the P210 bcr/abl oncogene product of the Philadelphia chromosome. Proc. Natl. Acad. Sci. U.S.A. 84:6558‐6562.
   McLaughlin, J., Chianese, E., and Witte, O.N. 1989. Alternative forms of the bcr‐abl oncogene have quantitatively different potencies for stimulation of immature lymphoid cells. Mol. Cell Biol. 9:1866‐1874.
   Miles, C., Sanchez, M.J., Sinclair, A., and Dzierzak, E. 1997. Expression of the Ly‐6E.1 (Sca‐1) transgene in adult hematopoietic stem cells and the developing mouse embryo. Development 124:537‐547.
   Million, R.P., Aster, J., Gilliland, D.G., and Van Etten, R.A. 2002. The Tel‐Abl (ETV6‐Abl) tyrosine kinase, product of complex (9;12) translocations in human leukemia, induces distinct myeloproliferative disease in mice. Blood 99:4568‐4577.
   Naviaux, R.K., Costanzi, E., Haas, M., and Verma, I.M. 1996. The pCL vector system: Rapid production of helper‐free, high‐titer, recombinant retroviruses. J. Virol. 70:5701‐5705.
   Neering, S.J., Bushnell, T., Sozer, S., Ashton, J., Rossi, R.M., Wang, P.Y., Bell, D.R., Heinrich, D., Bottaro, A., and Jordan, C.T. 2007. Leukemia stem cells in a genetically defined murine model of blast crisis CML. Blood 110:2578‐2585.
   Ory, D.S., Neugeboren, B.A., and Mulligan, R.C. 1996. A stable human‐derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. U.S.A. 93:11400‐11406.
   Parganas, E., Wang, D., Stravopodis, D., Topham, D.J., Marine, J.C., Teglund, S., Vanin, E.F., Bodner, S., Colamonici, O.R., van Deursen, J.M., Grosveld, G., andIhle, J.N. 1998. Jak2 is essential for signaling through a variety of cytokine receptors. Cell 93:385‐395.
   Pear, W.S., Nolan, G.P., Scott, M.L., and Baltimore, D. 1993. Production of high‐titer helper‐free retroviruses by transient transfection. Proc. Natl. Acad. Sci. U.S.A. 90:8392‐8396.
   Pear, W.S., Miller, J.P., Xu, L., Pui, J.C., Soffer, B., Quackenbush, R.C., Pendergast, A.M., Bronson, R., Aster, J.C., Scott, M.L., and Baltimore, D. 1998. Efficient and rapid induction of a chronic myelogenous leukemia‐like myeloproliferative disease in mice receiving P210 bcr/abl‐transduced bone marrow. Blood 92:3780‐3792.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Randall, T.D. and Weissman, I.L. 1997. Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5‐fluorouracil treatment. Blood 89:3596‐3606.
   Rosenberg, N. and Baltimore, D. 1976. A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia virus. J. Exp. Med. 143:1453‐1463.
   Roumiantsev, S., de Aos, I., Varticovski, L., Ilaria, R.L., and Van Etten, R.A. 2001. The Src homology 2 domain of Bcr/Abl is required for efficient induction of chronic myeloid leukemia‐like disease in mice but not for lymphoid leukemogenesis or activation of phosphatidylinositol 3‐kinase. Blood 97:4‐13.
   Roumiantsev, S., Krause, D.S., Neumann, C.A., Dimitri, C.A., Asiedu, F., Cross, N.C., and Van Etten, R.A. 2004. Distinct stem cell myeloproliferative/T lymphoma syndromes induced by ZNF198‐FGFR1 and BCR‐FGFR1 fusion genes from 8p11 translocations. Cancer Cell 5:287‐298.
   Smith, K.M., Yacobi, R., and Van Etten, R.A. 2003. Autoinhibition of Bcr‐Abl through its SH3 domain. Mol. Cell. 12:27‐37.
   Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
   Stocking, C., Kollek, R., Bergholz, U., and Ostertag, W. 1985. Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 82:5746‐5750.
   Swift, S., Lorens, J., Achacoso, P., and Nolan, G.P. 1999. Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell–based systems. Curr. Protoc. Immunol. 10.17.14‐10.17.29.
   Van Etten, R.A. 2001a. Models of chronic myeloid leukemia. Curr. Oncol. Rep. 3:228‐237.
   Van Etten, R.A. 2001b. Retroviral transduction models of Ph+ leukemia: Advantages and limitations for modeling human hematological malignancies in mice. Blood Cells Mol. Dis. 27:201‐205.
   Van Etten, R.A. 2002. Studying the pathogenesis of BCR‐ABL+ leukemia in mice. Oncogene 21:8643‐8651.
   Van Etten, R.A. and Shannon, K.M. 2004. Focus on myeloproliferative diseases and myelodysplastic syndromes. Cancer Cell 6:547‐552.
   Vardiman, J.W., Harris, N.L., and Brunning, R.D. 2002. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 100:2292‐2302.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Voytas, D. and Ke, N. 1999. Detection and quantitation of radiolabeled proteins and DNA in gels and blots. Curr. Protoc. Mol. Biol. 48:A.3A.1‐A.3A.10.
   Wernig, G., Mercher, T., Okabe, R., Levine, R.L., Lee, B.H., and Gilliland, D.G. 2006. Expression of Jak2V617F causes a polycythemia vera‐like disease with associated myelofibrosis in a murine bone marrow transplant model. Blood 107:4274‐4281.
   Wolff, N.C. and Ilaria, R.L.J. 2001. Establishment of a murine model for therapy‐treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571. Blood 98:2808‐2816.
   Xiao, S., Nalabolu, S.R., Aster, J.C., Ma, J., Abruzzo, L., Jaffe, E.S., Stone, R., Weissman, S.M., Hudson, T.J., and Fletcher, J.A. 1998. FGFR1 is fused with a novel zinc‐finger gene, ZNF198, in the t(8;13) leukaemia/lymphoma syndrome. Nat. Genet. 18:84‐87.
   Zaleskas, V.M., Krause, D.S., Lazarides, K., Patel, N., Hu, Y., Li, S., and Van Etten, R.A. 2006. Molecular Pathogenesis and Therapy of Polycythemia Induced in Mice by JAK2 V617F. PLoS ONE 1:e18.
   Zhang, X. and Ren, R. 1998. Bcr‐Abl efficiently induces a myeloproliferative disease and production of excess interleukin‐3 and granulocyte‐macrophage colony‐stimulating factor in mice: A novel model for chronic myelogenous leukemia. Blood 92:3829‐3840.
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