Blood Cytokines for In Utero Bone Marrow Transplantation

Human bone marrow can be transplanted into sheep fetuses when administered in utero. Upon birth of the sheep, the human hematopoietic stemcells produce differentiated human blood cells in the lymphoid erythroid and myeloid lineages for at least two years. Thus, the sheep/human chimera is an important clinical model for human stem cell (HSC) transplantation. The lack of recombinant sheep growth factors poses a major limitation to optimizing the growth of human cells in sheep transplants. One would like to manipulate blood levels of erythropoietin (Epo) to experimentally control red blood cell proliferation and differentiation; however, substantial quantities of pure sheep Epo are not available for injection. Readily available recombinant sheep Epo would contribute significantly to hemopoietic reconstitution studies in this valuable transplantation model. Since our group has considerable experience with the molecular genetics of Epo and its receptor, we will test the feasibility of cloning sheep Epo by determining the optimal probe available for detecting sheep Epo sequences, obtaining genomic clone of the sheep Epo gene and partial sequenced for cDNA cloning, identifying tissues expressing high levels of Epo mRNA (fetal liver and kidney, adult anemic kidney), and generating cDNA for PCR or expression-library cloning.