Genetic Reconstitution of Bone Marrow for the Study of Signal Transduction Ex Vivo

Introducing genes into cells by retroviral transduction has greatly increased the abil-ity to study signal transduction pathways in primary cells. Retroviral transduction has proven to be an efficient method to express genes of interest in cells that are difficult to manipulate using standard transfection techniques. This technology also can be coupled with classic protocols for generating bone marrow chimeras. Murine bone marrow cells can be infected with a retrovirus expressing wild-type or mutant forms of a gene of interest and subsequently transplanted into irradiated recipient hosts. The requirement for a gene of interest in hematopoietic cell development, as well as its role in specific signal transduction pathways, can then be studied. This chapter provides protocols for the production of high-titer replication-incompetent retrovirus, retroviral infection of murine bone marrow, the generation of bone marrow chimeras, and analysis of chimeras by flow cytometry.