Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression.
Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression.
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验证使用含有巨细胞病毒启动子的人工线粒体报告 DNA 载体进行线粒体转基因表达。
DOI：
10.1016/j.biomaterials.2017.05.016
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Harashima H.
中科院分区：
文献类型：
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作者：
Yamada Y;Ishikawa T;Harashima H.
线粒体有自己的独立于核系统的基因表达系统，并与核协同控制细胞功能。虽然已经报道了一些实现核转基因表达的有用技术，但只有几种技术专注于线粒体。在本研究中，我们验证了带有病毒启动子的人工线粒体DNA载体对线粒体转基因表达的有效性。我们设计并构建了含有巨细胞病毒启动子的pCMV-mtLuc(CGG)，以及带有NanoLuc(Nluc)荧光素酶基因的人工线粒体基因组，该基因记录了线粒体密码子系统的调节。Nluc荧光素酶活性测定表明，pCMV-mtLuc(CGG)能在人HeLa细胞中高效表达Nluc荧光素酶蛋白。此外，我们使用Mito-Porter系统优化了pCMV-mtLuc(CGG)的线粒体转染，Mito-Porter系统是一种基于脂质体的线粒体转移膜融合载体。结果发现，经Kala(阳离子两亲性细胞穿透肽)修饰的Mito-Porter载体pCMV-mtLuc(CGG)具有较高的线粒体转基因表达。开发的线粒体转基因表达系统为纳米科学和纳米技术领域通过调控线粒体功能来控制细胞内微环境提供了一种潜在的有用工具，并有望开辟更多的创新研究领域。
Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial deliveryviamembrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironmentviathe regulation of mitochondrial function and promises to open additional innovative research fields of study.