The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny
The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny
复制标题
DOI：
10.1073/pnas.100471497
复制
发表时间：
2000-05-23
影响因子：
11.1
通讯作者：
Marth, JD
中科院分区：
文献类型：
--
作者：
Shafi, R;Lyer, SPN;Marth, JD
核和细胞质蛋白糖基化是真核细胞中广泛且可逆的翻译后修饰。 O-GlcNAc 转移酶 (OGT) 催化将 N-乙酰氨基葡萄糖 (GlcNAc) 添加到丝氨酸和苏氨酸上而发生细胞内糖基化。细胞内蛋白质的这种“O-GlcNAc酰化”可以发生在磷酸化位点上，并且与控制基因转录、神经丝组装和糖尿病酸性神经系统疾病的出现有关。为了研究 OGT 体内功能，我们在男性胚胎干细胞中使用了基因靶向方法。我们发现 OCT 诱变需要一种保留完整 OGT 基因的策略，如使用 Cre-loxP 重组来完成的，因为 OGT 基因的缺失会导致胚胎干细胞活力丧失。 OGT 基因的单拷贝存在于雄性基因组中，在小鼠跨越标记 DxMit41 和 DxMit95 中位于靠近 D 区着丝粒的 X 染色体上，在人类中则位于 Xq13（与神经系统疾病相关的区域）。小鼠中的 OCT RNA 表达在大多数细胞类型中相对较高，但在胰腺中的水平较低。小鼠种系中 OGT 等位基因与卵母细胞中 ZP3-Cre 重组的分离表明，完整的 OGT 等位基因是完成胚胎发生所必需的。这些研究说明了条件基因靶向方法在重要性连锁基因的诱变和研究中的必要性，并表明 OGT 参与细胞内糖基化对于胚胎干细胞活力和小鼠个体发育至关重要。
Nuclear and cytoplasmic protein glycosylation is a widespread and reversible posttranslational modification in eukaryotic cells. Intracellular glycosylation by the addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalyzed by the O-GlcNAc transferase (OGT). This "O-GlcNAcylation" of intracellular proteins can occur on phosphorylation sites, and has been implicated in controlling gene transcription, neurofilament assembly, and the emergence of diabetes acid neurologic disease. To study OGT function in vivo, we have used gene-targeting approaches in male embryonic stem cells. We find that OCT mutagenesis requires a strategy that retains an intact OGT gene as accomplished by using Cre-loxP recombination, because a deletion in the OGT gene results in loss of embryonic stem cell viability. A single copy of the OGT gene is present in the male genome and resides on the X chromosome near the centromere in region D in the mouse spanning markers DxMit41 and DxMit95, and in humans at Xq13, a region associated with neurologic disease. OCT RNA expression in mice is comparably high among most cell types, with lower levels in the pancreas. Segregation of OGT alleles in the mouse germ line with ZP3-Cre recombination in oocytes reveals that intact OGT alleles are required for completion of embryogenesis. These studies illustrate the necessity of conditional gene-targeting approaches in the mutagenesis and study of essential sex-linked genes, and indicate that OGT participation in intracellular glycosylation is essential for embryonic stem cell viability and for mouse ontogeny.