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

转运系统x_c^-介导胞外半胱氨酸与胞内谷氨酸之间的交换转运。该系统由XCT和4F2hc两种蛋白组成，前者被认为是实际的转运蛋白。在本项目中，我们试图建立XCT基因敲除(XCT^&lt；-/-&gt；)小鼠，以阐明系统x_c^-在整个小鼠中的生理和病理作用。结果如下：1.成功地制备了XCT^&lt；-/-&gt；小鼠。通过聚合酶链式反应和基因组Southern杂交分析鉴定出XCT基因靶向破坏的纯合子小鼠。这些小鼠体内通过系统x_c^-转运半胱氨酸的活性完全丧失。XCT^&lt；-/-&gt；小鼠直到出生六个月后，外表和生育能力都很健康。显微镜下，在测试的任何器官中都没有发现异常，在血液学检查中，这些小鼠也没有发现异常。我们检测了血浆中的氨基酸。XCT^&lt；-/-&gt；小鼠血浆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明显差异。XCT^&lt；-/-&gt；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^&lt；-/-&gt；小鼠体内氧化还原失衡。XCT^&lt；-/-&gt；小鼠胚胎成纤维细胞在常规培养液中不能存活。在培养过程中，XCT^&lt；-/-&gt；小鼠细胞中的GSH急剧下降，这可能导致细胞死亡。在氧化应激或炎性刺激条件下，XCT^&lt；-/-&gt；与野生型小鼠的存活率无明显差异。我们研究了出生六个月以上的XCT^&lt；-/-&gt；小鼠的表型变化。然而，由于时间的限制，实验还没有完成。

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.