FES-Cre targets phosphatidylinositol glycan class A (PIGA) inactivation tohematopoietic stem cells in the bone marrow

A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIG A) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked prote ins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the muta tion occurs in a hematopoietic stein cell. In transgenic mice, germline tra nsmission of an inactive Papa gene is embryonic lethal. To inactivate the m urine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the Human c-fes gene. FES-Cre ina ctivated PIGA in hematopoietic cells of puce carrying a floxed Piga allele (LF mice). PIGA(-) cells were found in all hematopoietic lineages of defini tive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA(-) blood cells. The loss of GPI-lin ked proteins occurred mainly in c-kit(+)CD34(+)Lin(-) progenitor cells befo re the CFU-GEMM stage. Using bone marrow reconstitution experiments with pu rified PIGA- cells we demonstrate that LF mice have long-term bone marrow r epopulating cells that lack GPI-linked proteins, indicating that recombinat ion of the floxed Piga allele occurs in the hematopoietic stein cell.