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

我们发现，用一种抗肿瘤双插剂二元石治疗，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。由此产生的小鼠mtDNA-less （rho^0）细胞系通过rho^0细胞与小鼠脑突触体（代表从神经细胞中分离出来的突触末梢）融合，成功地用于捕获活神经细胞的mtDNA到分裂培养细胞中。无论mtDNA是来自年轻小鼠还是老年小鼠，具有神经元mtDNA的杂交克隆均获得了类似的线粒体翻译活性恢复，这表明至少线粒体基因组缺陷与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，我们可以捕获非常少量的常见5823 bp缺失突变体mtDNA (DELTAmtDNA^<5823>)，通过PCR在杂交克隆中检测到。由于大规模缺失的突变体mtDNA的数量预计会在杂交体的长期培养过程中增加，因此这些细胞应该可用于建立含有缺失突变体mtDNA的小鼠。虽然我们不能完成这个项目的主要目的，但是我们在rho^0小鼠细胞的分离上的成功将引导我们分离mtDNA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.