Construction of transgenic mice into which mutant mitochondrial DNA is artificially introduced.

人工引入突变线粒体 DNA 的转基因小鼠的构建。

• 批准号: 04557012
• 负责人: OHTA Shigeo
• 金额: $ 11.84万
• 依托单位: Instute of Gerontology, Nippon Mrdical School (1994)Jichi Medical University (1992-1993)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04557012/
• 关键词: Mitochondria; Transgenic mouse; Cybrid; Respiratory chain; particle gun; Cell fusion; Mitochondrial encephalomyopathy; Cardiomyopathy; タンパク質合成; Alpers病; 老化; パーラィクルガン; ミトコンドリアDNA; ミトコンドリア胸筋症; 遺伝子病; 遺伝子導入; 金属粒子; 遺伝子銃

mtDNA with a point mutation in the tRNA^<Ile> gene at nucleotide position 4269 found in a patient with fatal cardiomyopathy and mtDNA with a point mutation in the tRNA^<Arg> gene at 10410 found in a patient with Alpers disease were transferred cytoplasmically to rhoﾟ HeLa cells (HeLa cells lacking mtDNA) to determine whether these novel mtDNA mutations in the tRNA genes are responsible for the defects in mitochondrial respiration function observed in these diseases. Cybrid clones (clones of rhoﾟ HeLa cells with mtDNA from the patients) were isolated, and respiratory function and morphology of the mitochondria of the cybrid clones containing wild-type mtDNA and mutant mtDNA predominantly were compared. The results showed that accumulation of mutant mtDNA at 4269 alone without defects in the nuclear genome was sufficient to produce a disease phenotype, while mutant mtDNA at 10410 was not related to pathogenesis and reflected one of the rare polymorphic sites of human mtDNA.Moreover, we found that mitochondria in living cells were significantly swollen only when they contained predominantly the pathogenic mutant mtDNA,suggesting that the functional abnormalitiy of mitochondria induced by pathogenic mtDNA mutations in tRNA genes is always associated with their swollen structure.

将<Ile>在患有致命性心肌病的患者中发现的在tRNA 4基因的核苷酸位置4269处具有点突变的mtDNA和在<Arg>患有Alpers病的患者中发现的在tRNA 4基因的10410处具有点突变的mtDNA通过细胞质转移到rho-HeLa细胞（缺少mtDNA的HeLa细胞）中，以确定这些在tRNA 4基因中的新的mtDNA突变是否是在这些疾病中观察到的线粒体呼吸功能缺陷的原因。分离胞质杂交体克隆（rho-HeLa细胞与来自患者的mtDNA的克隆），并比较主要含有野生型mtDNA和突变型mtDNA的胞质杂交体克隆的呼吸功能和线粒体形态。结果表明，在核基因组中没有缺陷的mtDNA 4269位突变体单独积累就足以产生疾病表型，而mtDNA 10410位突变体与发病无关，反映了人类mtDNA的一种罕见多态性位点。此外，我们发现，活细胞中的线粒体只有在主要含有致病突变体mtDNA时才显著肿胀，提示致病性mtDNA tRNA基因突变引起的线粒体功能异常往往与线粒体肿胀结构有关。

项目成果

Kawashima, S., Ohta, S., Kagawa, Y., Yoshida, M., Nishizawa, M.: "Widespread tissue distribution of multiple mitochondrial DNA deletions in familial mitochondrial myopathy" Muscle and Nerve. 17. 741-746 (1994)

Kawashima, S.、Ohta, S.、Kakawa, Y.、Yoshida, M.、Nishizawa, M.：“家族性线粒体肌病中多个线粒体 DNA 缺失的广泛组织分布”肌肉和神经。

Matsuda, C., Endo, H., Ohta, S.and Kagawa, Y.: "Gene structure of human mitochondrial ATP synthase g-subunit ; Tissue specificity produced by RNA splicing.categorization of patients in Long-Ter" J.Biol.Chem.268. 24950-24958 (1993)

Matsuda, C.、Endo, H.、Ohta, S. 和 Kakawa, Y.：“人线粒体 ATP 合酶 g 亚基的基因结构；RNA 剪接产生的组织特异性。长期患者的分类” J.Biol

Hayashi,J-I.,他: "Accumulation of mtDNA with a mutaion at position 3271 in tRNA-Leu(UUR)gene introduced from a MELAS patient to HeLa cells lacking mtDNA results in progressive inhibition of mitochondrial respiratory function" Biochem Biophys.Res.Commun.197

Hayashi, J-I. 等人：“从 MELAS 患者引入的 tRNA-Leu(UUR) 基因中 3271 位突变的 mtDNA 累积到缺乏 mtDNA 的 HeLa 细胞中，导致线粒体呼吸功能进行性抑制” Biochem Biophys.Res .通讯.197

Kabayashi,Y.: "The mutant mitochondrial genes in mitochondrial myopathy,encepholopathy,lactic acidosis and stroke-like episodes (MELAS)were selectively amplified through generations." J.Inher.Metab.Dis.15. 803-808 (1992)

Kabayashi,Y.：“线粒体肌病、脑病、乳酸性酸中毒和中风样发作 (MELAS) 中的突变线粒体基因经过几代人选择性扩增。”

Shiraiwa N., Ishii A., Iwamoto H., Mizusawa H., Kagawa Y.and Ohta S.: "Content of mutant mitochondrial DNA and organ-dysfunction in a patient with a MELAS-subgroup of mitochondrial encephalomyopathies." J.Neurol.Sci.120. 174-179 (1993)

Shiraiwa N.、Ishii A.、Iwamoto H.、Mizusawa H.、Kakawa Y.和 Ohta S.：“线粒体脑肌病 MELAS 亚组患者中突变线粒体 DNA 的含量和器官功能障碍。”