Assessment of multi-organ system engraftment by genotypic typing using restriction fragment-length polymorphisms and by phenotypic typing using a microcytotoxicity assay.
We report the first application of Southern blotting techniques for the quantitative assessment of the donor or host origin of cell populations present in recipients of allogeneic or sex-mismatched syngeneic murine donor marrow grafts. The sensitivity of this assay system was noted to 1 to 5% for detection of a minor cell population by using cDNA probes that hybridize to single-copy sequences in the murine genome. The use of probes that generate distinguishing autoradiographic patterns due to strain-specific genomic sequence variations obviates the need for retroviral vector transfections (which potentially skew engraftment quantitation). Southern blotting analysis has provided definitive engraftment data in multiple cell populations isolated from both short-term and long-term allogeneic and syngeneic radiation chimeras. In contrast, H-2 typing in a microcytotoxicity assay, a standard typing technique for allogeneic murine donor cell engraftment, was noted to be less sensitive than Southern blotting. This occurred particularly in selected cell populations, in ill-appearing recipients, and in the early post-BMT period. Furthermore, because H-2 typing is a phenotypic assay, the results may be substantially influenced by the passive cell surface acquisition of host H-2 antigens, a process that is not evident with the use of genotyping techniques. Our results establish the superiority of Southern blotting techniques for the quantitation of donor cell engraftment and demonstrate the potential of this methodology when low-level detection of engrafted donor or residual host cells is of critical physiologic importance.
D000818:Animals; D016026:Bone Marrow Transplantation; D003601:Cytotoxicity Tests, Immunologic; D006183:H-2 Antigens / Q000032:analysis; D051379:Mice; D012150:Polymorphism, Restriction Fragment Length; D011828:Radiation Chimera; D012730:Sex Chromosomes; D013997:Time Factors
D006183:H-2 Antigens