A KNOCK-OUT MODEL OF PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA - PIG-A(-) HEMATOPOIESIS IS RECONSTITUTED FOLLOWING INTERCELLULAR TRANSFER OF GPI-ANCHORED PROTEINS

We created a ''knock-out'' embryonic stem cell via targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene, resulting in loss of expression of cell surface glycosyl phosphatidylinositol-anch ored proteins and reproducing the mutant phenotype of the human diseas e paroxysmal nocturnal hemoglobinuria. Morphogenesis of Pig-a(-) embry oid bodies (EB) in vitro was grossly aberrant and, unlike EB derived f rom normal embryonic stem cells, Pig-A(-) EB produced no secondary hem atopoietic colonies. Chimeric EB composed of control plus Pig-A(-) cel ls, however, appeared normal, and hematopoiesis from knock-out cells w as reconstituted. Transfer in situ of glycosyl phosphatidylinositol-an chored proteins from normal to knock-out cells was demonstrated by two -color fluorescent analysis, suggesting a possible mechanism for these functional effects. Hematopoietic cells with mutated PIG-A genes in h umans with paroxysmal nocturnal hemoglobinuria may be subject to compa rable pathophysiologic processes and amenable to similar therapeutic p rotein transfer.