Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal mitochondrial DNA with deletion mutations
Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal mitochondrial DNA with deletion mutations
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DOI：
10.1074/jbc.272.24.15510
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发表时间：
1997-06-13
影响因子：
4.8
通讯作者：
Hayashi, JI
中科院分区：
文献类型：
--
作者：
Inoue, K;Ito, S;Hayashi, JI
为了分离小鼠无 mtDNA (rho(0)) 细胞系，我们寻找了各种有望降低 mtDNA 含量的抗线粒体药物，并发现用双特卡林（一种抗肿瘤双嵌入剂）治疗对于完全排除我们测试的所有小鼠细胞系中的 mtDNA 非常有效。所得的rho(0)小鼠细胞通过将rho(0)细胞与小鼠脑突触体融合，成功地将活体神经细胞的mtDNA捕获到分裂的培养细胞中，突触体代表从神经细胞中分离出的突触末端。获得神经元mtDNA后，所有cybrid克隆都类似地恢复了线粒体翻译活性，无论mtDNA是来自年轻的还是年老的 小鼠，因此至少表明线粒体基因组的缺陷与在老年小鼠大脑中观察到的与年龄相关的线粒体功能障碍无关。此外，我们可以捕获极少量的常见5823碱基对缺失突变体mtDNA（Delta mtDNA（5823）），该突变体可通过cybrid中的聚合酶链反应检测到 克隆。由于在细胞杂种的长期培养过程中，大规模缺失的突变体 mtDNA 的数量预计会增加，因此这些细胞应该可用于建立含有缺失突变体 mtDNA 的小鼠。
For isolation of mouse mtDNA-less (rho(0)) cell lines, we searched for various antimitochondrial drugs that were expected to decrease the mtDNA content and found that treatment with ditercalinium, an antitumor bis-intercalating agent, was extremely effective for completely excluding mtDNA in all the mouse cell lines we tested. The resulting rho(0) mouse cells were successfully used for trapping the mtDNA of living nerve cells into dividing cultured cells by fusion of the rho(0) cells with mouse brain synaptosomes, which represent synaptic endings isolated from nerve cells, With neuronal mtDNA obtained, all of the cybrid clones restored mitochondrial translation activity similarly regardless of whether the mtDNA was derived from young or aged mice, thus at least suggesting that defects in mitochondrial genomes are not involved in the age-associated mitochondrial dysfunction observed in the brain of aged mice, Furthermore, we could trap a very small amount of a common 5823-base pair deletion mutant mtDNA (Delta mtDNA(5823)) that was detectable by polymerase chain reaction in the cybrid clones. As the amount of mutant mtDNA with large scale deletions was expected to increase during prolonged cultivation of the cybrids, these cells should be available for establishment of mice containing the deletion mutant mtDNA.