Platelets and Megakaryocytes

Volume 273 of the series Methods in Molecular Biology™ pp 381-396

Using Retroviruses to Express Genes in Primary Megakaryocyte Lineage Cells

  • Meenakshi GaurAffiliated withDepartments of Laboratory and Internal Medicine, University of California
  • , George J. MurphyAffiliated withDepartment of Genetics, Children’s Hospital, Harvard Medical School
  • , Jonathan FramptonAffiliated withDepartment of Anatomy, Birmingham University Medical School
  • , Andrew D. LeavittAffiliated withDepartments of Laboratory and Internal Medicine, University of California

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Megakaryocytes in adult human bone marrow are estimated to constitute approx 0.4% of the total marrow cells (1), and our experience suggests that fewer than 0.5% of low-density nucleated murine bone marrow cells express the megakaryocyte-lineage marker CD41 (integrin αIIb). Historically, the infrequent occurrence of megakaryocyte-lineage cells in bone marrow has been a significant obstacle to the procurement of primary megakaryocyte-lineage cells for biological studies. However, currently available conditions allow one to expand these cells in culture. In this chapter we describe protocols for using retroviruses to selectively infect early megakaryocyte-lineage cells and to infect mature megakaryocytes. The protocols allow one to study the effect of specific gene products on lineage development and biological functions of these primary cells. A basic understanding of retroviruses and the retrovirus life cycle is assumed (for review, see refs. 2,3).