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Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism >80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice