Generation and analysis of cystine/glutamate transporter gene-modified mouse

运输系统x_c^-介导细胞外胱氨酸和细胞内谷氨酸之间的交换运输。该系统由两种蛋白质组成，xCT和4F2hc，前者被认为是真正的转运蛋白。本项目尝试构建xCT基因敲除（xCT^<-/->）小鼠，以阐明系统x_c^-在全鼠中的生理病理作用。研究结果如下：1。xCT^<-/->小鼠已成功生产。通过PCR和基因组Southern blot分析鉴定了靶向破坏xCT基因的小鼠纯合子。通过x_c^-系统的胱氨酸运输活性在这些小鼠中完全丧失。直到出生6个月后，小鼠的外观和生育能力都很健康。显微镜下未发现任何器官异常，血液学检查也未发现异常。我们检查了血浆氨基酸。xCT^<-/->小鼠的半胱氨酸浓度显著高于野生型小鼠，而血浆半胱氨酸浓度无显著差异。xCT^<-/->小鼠血浆谷胱甘肽（GSH）水平低于野生型小鼠。这些结果表明xCT^<-/->小鼠存在氧化还原失衡。xCT^<-/->小鼠的胚胎成纤维细胞在常规培养基中不能存活。xCT^<-/->小鼠细胞GSH在培养过程中急剧下降，可能导致细胞死亡。在氧化应激和炎症刺激条件下，xCT^<-/->小鼠的存活率与野生型小鼠无明显差异。我们研究了xCT^<-/->小鼠出生后6个月的表型变化。然而，由于时间的限制，实验一直没有完成。

Transport system x_c^- mediates the exchange transport between extracellular cystine and intracellular glutamate. This system is composed of two proteins, xCT and 4F2hc, and the former is thought to be the actual transporter. In this project we have tried to produce xCT gene-knockout (xCT^<-/->) mouse to elucidate the physiological and pathological role of system x_c^- in whole mouse. The results are as follows :1. xCT^<-/-> mice have been successfully produced. Mice homozygous for the targeted disruption of the xCT gene were identified by PCR and genomic Southern blot analysis. Activity of cystine transport via system x_c^- was completely lost in these mice.2. xCT^<-/-> mice, until six months after birth, were apparently healthy in appearance and fertile.Microscopically no abnormalities were found in any of the organs tested and, in hematological investigations, no abnormalities were found in these mice.3. We have examined plasma amino acids. Cystine concentration in xCT^<-/-> mice is significantly higher than in wild type mice while there was no difference in plasma cysteine concentration. Plasma glutathione (GSH) level in xCT^<-/-> mice was lower than that in wild type mice. These results suggest redox imbalance in xCT^<-/-> mice.4. The embryonic fibroblasts derived from xCT^<-/-> mice failed to survive in routine culture medium. GSH in the cells from xCT^<-/-> mice drastically decreased during the culture and this may cause cell death.5. We could not find any difference between the survival rate of xCT^<-/-> and wild type mice under conditions of oxidant stress or of inflammatory stimulus.6. We have investigated a phenotype change of xCT^<-/-> mice over six month after birth. However, the experiments have not been completed due to a restriction by time.