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Assessment of Lymphocyte Development in Radiation Bone Marrow Chimeras

Gerald J. Spangrude1

1University of Utah, Salt Lake City, Utah

Publication Name: 
Current Protocols in Immunology
Unit Number: 
Unit 4.6
DOI: 
10.1002/0471142735.im0406s81
Online Posting Date: 
May, 2008
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Abstract

Transplantation of marrow between mouse strains congenic for CD45 after lethal irradiation establishes hematopoiesis driven by genetically marked cells in recipient animals. After several weeks, peripheral blood or primary and secondary lymphoid organs of transplant recipients can be evaluated for the presence of donor-derived cells. Two- or three-color flow cytometry can be used to identify the progeny of transplanted cells, to document their cell-surface phenotypes, and to follow development of T, B, and myeloid lineages in vivo. Curr. Protoc. Immunol. 81:4.6.1-4.6.9. © 2008 by John Wiley & Sons, Inc.

Keywords: bone marrow transplantation; green fluorescent protein; radiation; chimerism analysis; animal husbandry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol: Preparation of Radiation Chimeras
  • Support Protocol 1: Determination of Radiation Doses
  • Support Protocol 2: Detection of Donor-Derived Cells
  • Support Protocol 3: Screening for Pseudomonas Aeruginosa in Mouse Colonies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol: Preparation of Radiation Chimeras

 Materials
  • Bone marrow recipient animals: six to twelve C57BL/6 females, 8 to 12 weeks old (The Jackson Laboratory)
  • 2 mg/ml neomycin sulfate (Sigma; prepare in tap water)
  • Bone marrow donor animal: congenic B6.SJL-Ptprca Pep3b/BoyJ or transgenic B6-Tg(ACTb-EGFP) female, <6 months old (The Jackson Laboratory)
  • Control bone marrow donor animal; C57BL/6 female, <6 months old (The Jackson Laboratory)
  • Hanks’ balanced salt solution (HBSS; appendix 2A) or phosphate-buffered saline (PBS; appendix 2A)
  • X-ray machine or cesium source for irradiation
  • Additional reagents and equipment for determination of radiation dose to be used to prepare radiation chimera (Support Protocol 1), animal euthanasia (unit 1.8), preparing bone marrow suspensions (unit 6.4), counting cells (appendix 3A), intravenous injections (unit 1.6), and detection of donor-derived cells in radiation chimeras (Support Protocol 2)

Support Protocol 1: Determination of Radiation Doses

 Materials
  • Bone marrow recipient animals: 16 to 24 C57BL/6 females, 8 to 12 weeks old (The Jackson Laboratory)
  • Bone marrow donor animal: congenic B6.SJL-Ptprca Pep3b/BoyJ or transgenic B6-Tg(ACTb-EGFP) female, <6 months old (The Jackson Laboratory)
  • Hanks’ balanced salt solution (HBSS; appendix 2A) or phosphate-buffered saline (PBS; appendix 2A)
  • 2 mg/ml neomycin sulfate (Sigma; prepare in tap water)
  • X-ray machine or cesium source for irradiation
  • Additional reagents and equipment for animal euthanasia (unit 1.8), preparing bone marrow suspensions (unit 6.4), counting cells (appendix 3), intravenous injections (unit 1.6), and detection of donor-derived cells in radiation chimeras (Support Protocol 2)

Support Protocol 2: Detection of Donor-Derived Cells

 Materials
  • Irradiated and reconstituted mice (Basic Protocol or Support Protocol 1)
  • Phosphate-buffered saline (PBS; appendix 2A) containing 3 mg/ml EDTA plus 1 U/ml heparin, pH 7.2
  • 20 mg/ml dextran T-500 (GE Healthcare) in PBS
  • ACK lysing buffer (see recipe)
  • Hanks’ balanced salt solution (HBSS; appendix 2A) containing 5% (v/v) newborn calf serum (CS)
  • Fluorescein-labeled MAb A20.1 specific for the CD45a (Ly5.1) allele (eBiosciences)
  • Phycoerythrin-labeled MAbs to detect lymphoid-specific antigens of interest (e.g., CD3, CD4, CD8, CD19) (eBiosciences)
  • 5-ml centrifuge tubes
  • Refrigerated centrifuge
  • Additional reagents and equipment for collecting peripheral blood (unit 1.7), immunofluorescent staining (unit 5.3), and flow cytometry (unit 5.4)

Support Protocol 3: Screening for Pseudomonas Aeruginosa in Mouse Colonies

 Materials
  • Irradiated mouse to be tested (Basic Protocol or Support Protocol 1)
  • Luria-Bertani (LB) broth, Miller formulation (BD Difco)
  • Cetrimide agar plates (Pseudosel agar, BBL or BD Difco) or Pseudomonas Isolation Agar (BD Difco)
  • Sterile cotton-tipped applicator sticks
  • Sterile microbiological culture tubes
  • Additional reagents and equipment for animal restraint (unit 1.3) and animal identification (unit 1.5)
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Literature Cited

Literature Cited
    Becker, A.J., McCulloch, E.A., and Till, J.E. 1963. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197: 452-454.
    Dominici, M., Tadjali, M., Kepes, S., Allay, E.R., Boyd, K., Ney, P.A., Horwitz, E., and Persons, D.A. 2005. Transgenic mice with pancellular enhanced green fluorescent protein expression in primitive hematopoietic cells and all blood cell progeny. Genesis 42: 17-22.
    Goldschneider, I., Komschlies, K.L., and Greiner, D.L. 1986. Studies of thymocytopoiesis in rats and mice. I. Kinetics of appearance of thymocytes using a direct intrathymic adoptive transfer assay for thymocyte precursors. J. Exp. Med. 163: 1-17.
    Jordan, C.T. and Lemischka, I.R. 1990. Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Dev. 4: 220-232.
    Krause, D.S., Theise, N.D., Collector, M.I., Henegariu, O., Hwang, S., Gardner, R., Neutzel, S., and Sharkis, S.J. 2001. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105: 369-377.
    McDougall, P.T., Wolf, N.S., Stenback, W.A., and Trentin, J.J. 1967. Control of Pseudomonas aeruginosa in an experimental mouse colony. Lab. Animal Care 17: 204-214.
    Micklem, H.S., Clarke, C.M., Evans, E.P., and Ford, C.E. 1968. Fate of chromosome-marked mouse bone marrow cells transfused into normal syngeneic recipients. Transplantation 6: 299-302.
    Shen, F.-W., Saga, Y., Litman, G., Freeman, G., Tung, J.-S., Cantor, H., and Boyse, E.A. 1985. Cloning of Ly-5 cDNA. Proc. Natl. Acad. Sci. U.S.A. 82: 7360-7363.
    Spangrude, G.J. and Scollay, R. 1990. Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes: Kinetics and phenotype of progeny. J. Immunol. 145: 3661-3668.
    Spangrude, G.J., Cho, S., Guedelhoefer, O., Vanwoerkom, R.C., and Fleming, W.H. 2006. Mouse models of hematopoietic engraftment: Limitations of transgenic green fluorescent protein strains and a high-performance liquid chromatography approach to analysis of erythroid chimerism. Stem Cells 24: 2045-2051.
 Key Reference
    Spangrude, G.J., Heimfeld, S., and Weissman, I.L. 1988. Purification and characterization of mouse hematopoietic stem cells. Science 241: 58-62.

Describes the use of the CD45 allelic system to track engraftment of nucleated cells in a radiation chimera model.

     
 
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