Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast
Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast
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DOI：
10.1007/s11095-011-0527-1
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发表时间：
2011-11-01
影响因子：
3.7
通讯作者：
Coutelle, Charles
中科院分区：
文献类型：
--
作者：
Bigger, Brian W.;Liao, Ai-Yin;Coutelle, Charles
人类线粒体基因传递载体的开发是治疗线粒体DNA突变引起的疾病的关键一步。虽然我们之前已经克隆了完整的小鼠线粒体基因组，并将其开发为线粒体基因治疗载体，但由于d -环和tRNA（Thr）基因的不稳定区域，人类线粒体基因组在大肠杆菌中被称为不可克隆。我们测试了在大肠杆菌和酿酒酵母中克隆人类线粒体DNA的多拷贝和单拷贝载体系统，包括转化相关的重组。人线粒体DNA在大肠杆菌中是不可克隆的，在任何条件下都不能保留在多拷贝或单拷贝载体中。然而，只要使用单拷贝着丝粒质粒，就有可能在酵母中克隆并稳定维持整个人类线粒体基因组。D-loop和tRNA（Thr）均稳定且未突变。这是克隆人类线粒体基因组的第一篇报道，也是开发人类线粒体基因治疗基因传递载体的第一步。
Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene.We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated.This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.