Analyzes of genes responsible for aging and the pathogenesis of diabetes by isolation of transgenic mice with pathogenic mtDNA mutation

我们发现，在我们测试的所有小鼠细胞系中，用抗肿瘤双嵌入剂二特卡林治疗完全排除mtDNA是非常有效的。通过Rho^0细胞与代表神经细胞突触末梢的小鼠脑突触小体的融合，成功地将小鼠mtDNA缺失(Rho^0)细胞系用于捕获活神经细胞的mtDNA以分裂培养细胞。无论线粒体DNA是来自年轻小鼠还是老年小鼠，带有神经元mtDNA的环体克隆都获得了类似的恢复的线粒体翻译活性，这表明至少线粒体基因组的缺陷与在老年小鼠脑中观察到的与年龄相关的线粒体功能障碍无关。此外，我们可以捕获一个非常少量的常见的5823bp的缺失突变mtDNA(DELTAmtDNA^；5823&gt；)，这是在环体克隆中可以检测到的。随着线虫培养时间的延长，大量缺失突变mtDNA的数量有望增加，这些细胞应可用于建立含有缺失突变mtDNA的小鼠。虽然我们未能达到本项目的主要目的，但我们成功分离Rho^0小鼠细胞将使我们获得mtDNA基因敲除小鼠。

We 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 mouse mtDNA-less (rho^0) cell lines were succesafully used for trapping 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. The cybrid clones with neuronal mtDNA obtained all restored mitochondrial translation activity similarly irrespective of whether the mtDNA was derived from young or aged mice, suggesting that at least 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 bp-deletion mutant mtDNA (DELTAmtDNA^<5823>) that was detectable by PCR 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.Although we could not accomplish the main purpose of this project, our sucesss for isolation of rho^0 mouse cells would lead us to the isolation of mtDNA knockout mice.