Isolation of mtDNA knock-out mice by introduction of disease-related mtDNA mutation

通过引入疾病相关 mtDNA 突变来分离 mtDNA 敲除小鼠

• 批准号: 06557040
• 负责人: HAYASHI Jun-ichi
• 金额: $ 10.11万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557040/
• 关键词: mtDNA; mitochondrial diseases; mtDNA inlroduction; mitochondrial KO mice; disease-model mice; マウスmtDNA; mtDNA突然変異; mtDNA欠損細胞; ミトコンドリア病; 病態モデルマウス; シナプトゾーム; 老化; 糖尿病; トランスジェニックマウス

For isolation of the mouse mtDNA-less (rho^0) cell lines, we searched for various anti-mitochondrial drugs which 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 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.

为了分离小鼠无线粒体DNA（rho^0）细胞系，我们寻找了各种预期会降低线粒体DNA含量的抗线粒体药物，并发现用抗肿瘤双嵌入剂ditercalinium处理对完全排除我们测试的所有小鼠细胞系中的线粒体DNA非常有效。通过将rho^0细胞与小鼠脑突触体（代表从神经细胞中分离的突触末梢）融合，所得到的rho^0小鼠细胞成功地用于将活神经细胞的mtDNA捕获到分裂的培养细胞中。具有神经元mtDNA的胞质杂交体克隆获得了所有恢复的线粒体翻译活性，无论mtDNA是来自年轻小鼠还是老年小鼠，这表明至少线粒体基因组中的缺陷不涉及在老年小鼠脑中观察到的与年龄相关的线粒体功能障碍。此外，我们可以捕获非常少量的常见的5823 bp缺失突变体mtDNA（DELTAmtDNA^<5823>），其可以通过PCR在胞质杂交体克隆中检测到。由于在胞质杂交体的长期培养期间，具有大规模缺失的突变mtDNA的量预计会增加，因此这些细胞应该可用于建立含有缺失突变mtDNA的小鼠。

项目成果

林純一: "ミトコンドリア病:ミトコンドリアの体細胞遺伝学" 最新医学. (印刷中). (1995)

Junichi Hayashi：“线粒体疾病：线粒体的体细胞遗传学”现代医学（出版中）。

Sadamitsu Asoh: "Expression of the apoptosis-mediator Fas is enhanced by dysfunctional mitochondria" J.Biochem.120. 600-607 (1996)

Sadamitsu Asoh：“线粒体功能障碍增强了凋亡介导 Fas 的表达”J.Biochem.120。

Hideki Kaneda: "Selective elimination of paternal mitochondrial DNA during mouse embryogenesis." Proc.Natl.Acad.Sci.USA. 92. 4542-4546 (1995)

Hideki Kaneda：“在小鼠胚胎发生过程中选择性消除父本线粒体 DNA。”

林 純一: "ヒトのミトコンドリアとミトコンドリアゲノムは機械的に連続した一つの単位として働く" 蛋白質 核酸 酵素. 40. 143-150 (1995)

Junichi Hayashi：“人类线粒体和线粒体基因组作为一个机械连续单位发挥作用”蛋白质核酸酶。 40. 143-150 (1995)

Kimiko Inoue: "Mutant mtDNA at 1555 A to G in 12S rRNA gene and hypersusceptibility of mitochondrial translation to streptomycin can be co-transferred to ρ^o HeLa cells" Biochem.Biophys.Res.Commun.223. 496-501 (1996)

Kimiko Inoue：“12S rRNA 基因中 1555 A 至 G 处的突变 mtDNA 和线粒体翻译对链霉素的超敏感性可以共同转移至 ρ^o HeLa 细胞”Biochem.Biophys.Res.Commun.223 (1996)。