Mitochondria Hub Regulation Center and Department of Anatomy and Cell Biology, Dong-A University College of Medicine, 3-1 Dongdaesin-dong, Seo-gu, Busan 602-714, Republic of Korea.
J Genet Genomics. 2011 Apr 20;38(4):173-9. doi: 10.1016/j.jgg.2011.03.005. Epub 2011 Mar 23.
Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.
由于技术困难，哺乳动物细胞中线粒体的遗传转化仍然是一个挑战。在本报告中，我们描述了我们试图使用基于药物抗性基因选择的工程化线粒体基因组来转化哺乳动物线粒体的尝试。由于标准的抗药性新霉素磷酸转移酶在靶向线粒体时会赋予对高浓度 G418 的抗性，因此我们生成了一种经重新编码的新霉素抗性基因，该基因使用哺乳动物线粒体遗传密码来指导该蛋白在线粒体中而不是在核中（线粒体版本）的合成。我们还生成了经重新编码的新霉素抗性基因的通用版本，该版本允许在线粒体和核中合成具有抗药性的蛋白。当我们通过电穿孔将这些整合到小鼠线粒体基因组克隆中的经重新编码的新霉素抗性基因转染到小鼠组织培养细胞中时，没有 DNA 构建体被递送到线粒体中。我们发现，通用版本的经重新编码的新霉素抗性基因在核中表达，从而赋予对 G418 选择的抗性，而基因的合成线粒体版本不会从核转化产生任何背景抗性细胞。随着精确的线粒体转化技术的发展，这些经重新编码的合成抗性基因可能成为选择线粒体遗传转化体的有用工具。
