USE OF ALLOGENEIC BONE-MARROW LABELED WITH NEOMYCIN RESISTANCE GENE TO EXAMINE BONE-MARROW-DERIVED CHIMERISM IN EXPERIMENTAL ORGAN-TRANSPLANTATION

Posttransplant infusion of viable donor bone marrow cells (DBMC) has b een shown in our previous studies to promote acceptance of incompatibl e kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes, In this no nhuman primate model, the infusion of the DBMC is requisite for the in duction of functional graft tolerance and specific MLR and CTLp unresp onsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain, Standard immunological and molecular tec hniques applied to this monkey model are unable to differentiate betwe en chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants, To distinguish chimeri sm due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neo(r) using the retroviral vector pHSG-Neo. Neo(r)-t ransduced BMC were infused into recipients approximately 2 wk after ki dney transplantation and treatment with rabbit antithymocyte globulin, No maintenance immunosuppressive drugs were given, Genomic DNA isolat ed from peripheral blood leukocytes was used to monitor the presence o f Neo(r)-positive cells. Tissue samples obtained at necropsy also were assessed for Neo(r)-positive chimeric cells, The presence of DBMC-der ived chimerism was assessed by polymerase chain reaction using Neo(r) sequence-specific primers (PCR-SSP), Chimerism was detectable in recip ient tissues at various times for up to 6 mo after DBMC infusion, Thes e studies using gene transduction methodology indicate that a stable g enetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a nonhuman primate model, This approach shou ld facilitate future studies in preclinical models to study the role a nd type of chimeric cell lineages in relation to functional allograft tolerance. (C) 1997 Elsevier Science Inc.