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

  • 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

  • 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

  • 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

  • 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

  • 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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