Nozaki M, Ohishi K, Yamada N, Kinoshita T, Nagy A, Takeda J
Department of Molecular Embryology, Research Institute for Microbial Diseases, Osaka University, Japan.
Lab Invest. 1999 Mar;79(3):293-9.
One mode used to link membrane proteins to a cell membrane is by means of a special glycolipid anchor termed glycosylphosphatidylinositol (GPI). Pig-a, an X-linked gene, is involved in the first step of GPI-anchor biosynthesis. Disruption of this gene causes cessation of GPI biosynthesis on the endoplasmic reticulum, thereby leading to the absence of GPI-anchored proteins on the cell surface. We have previously reported that mice with high chimerism was never obtained from Pig-a disrupted ES cells, suggesting that GPI-anchored protein(s) may have important roles for mouse development such that the absence of GPI-anchored proteins causes a lethal effect to mice. In this study, this lethal effect has been investigated by using a conditional approach to "knockout" the Pig-a gene. For this, mice harboring a Pig-a gene flanked by two loxP sites (Pig-aflox) were mated with hCMV-Cre transgenic mice, which express Cre recombinase before implantation. The allele disruptions were identified by PCR analysis of embryo yolk sac DNA. Embryos harboring a complete disruption of Pig-a gene ceased to develop beyond the ninth day of gestation. Female embryos in which one Pig-a allele was disrupted by Cre such that only half of the cells in the embryo proper did not express GPI-anchored proteins due to random X inactivation developed until 19 days post coitum (dpc), but showed abnormal phenotypes such as insufficient closure of neural tube and cleft palate. These data further highlight the importance of GPI-anchored proteins during mouse embryonic development.
一种将膜蛋白连接到细胞膜的方式是通过一种特殊的糖脂锚，称为糖基磷脂酰肌醇（GPI）。Pig-a是一个X连锁基因，参与GPI锚生物合成的第一步。该基因的破坏会导致内质网上GPI生物合成的停止，从而导致细胞表面缺乏GPI锚定蛋白。我们之前报道过，从Pig-a基因敲除的胚胎干细胞中从未获得高嵌合率的小鼠，这表明GPI锚定蛋白可能对小鼠发育具有重要作用，以至于缺乏GPI锚定蛋白会对小鼠产生致命影响。在本研究中，通过使用条件性方法“敲除”Pig-a基因来研究这种致命影响。为此，将携带两个loxP位点侧翼的Pig-a基因（Pig-aflox）的小鼠与hCMV-Cre转基因小鼠交配，后者在植入前表达Cre重组酶。通过对胚胎卵黄囊DNA进行PCR分析来鉴定等位基因的破坏情况。完全破坏Pig-a基因的胚胎在妊娠第九天之后停止发育。由于随机X染色体失活，其中一个Pig-a等位基因被Cre破坏，导致胚胎自身只有一半细胞不表达GPI锚定蛋白的雌性胚胎发育到交配后19天（dpc），但表现出异常表型，如神经管闭合不全和腭裂。这些数据进一步突出了GPI锚定蛋白在小鼠胚胎发育过程中的重要性。
