Keller P, Payne J L, Tremml G, Greer P A, Gaboli M, Pandolfi P P, Bessler M
Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
J Exp Med. 2001 Sep 3;194(5):581-9. doi: 10.1084/jem.194.5.581.
A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine 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 inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA(-) cells were found in all hematopoietic lineages of definitive 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-linked proteins occurred mainly in c-kit(+)CD34(+)Lin(-) progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA(-) cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.
X连锁的磷脂酰肌醇聚糖A类（PIGA）基因中的体细胞突变导致阵发性夜间血红蛋白尿患者血细胞上糖基磷脂酰肌醇（GPI）连接蛋白的缺失。由于所有血细胞谱系都可能受到影响，因此认为该突变发生在造血干细胞中。在转基因小鼠中，无活性的Piga基因的种系传递是胚胎致死性的。因此，为了在早期造血过程中使小鼠Piga基因失活，我们选择了使用Cre/loxP系统进行条件性基因失活。我们在人c-fes基因转录调控序列的控制下表达Cre重组酶。FES-Cre使携带floxed Piga等位基因的小鼠（LF小鼠）造血细胞中的PIGA失活。在定型造血而非原始造血的所有造血谱系中均发现了PIGA（-）细胞。它们在新生小鼠中的比例较低，但随后持续增加，首次产生几乎完全是PIGA（-）血细胞的小鼠个体。GPI连接蛋白的缺失主要发生在CFU-GEMM阶段之前的c-kit（+）CD34（+）Lin（-）祖细胞中。通过用纯化PIGA（-）细胞进行骨髓重建实验，我们证明LF小鼠具有缺乏GPI连接蛋白且具有长期骨髓重建能力的细胞，这表明floxed Piga等位基因的重组发生造血干细胞中。
