Transformation of isolated mammalian mitochondria by bacterial conjugation.
Transformation of isolated mammalian mitochondria by bacterial conjugation.
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DOI：
10.1093/nar/gni140
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发表时间：
2005-09-12
影响因子：
14.9
通讯作者：
Koob, Michael D
中科院分区：
文献类型：
--
作者：
Yoon, Young Geol;Koob, Michael D
我们已经开发了一种方法，将外源DNA分子转移到分离的哺乳动物线粒体使用细菌偶联。一般来说，我们通过(i)将DNA转移（oriT）序列插入DNA构建体中，（ii）将构建体转化为合适的大肠杆菌菌株，然后（iii）通过偶联将可动员的DNA引入线粒体来实现这一目标。我们通过将含有T7启动子序列的质粒DNA转移到我们设计的含有T7 RNA聚合酶的线粒体中来测试这种方法。在大肠杆菌与线粒体结合后，我们检测到从转移到线粒体的DNA构建体中有高水平的T7转录。这种在体外构建工程DNA并随后通过偶联转移到线粒体的方法为研究脊椎动物线粒体基因表达的许多方面提供了一个有吸引力的实验系统，并且是转化哺乳动物细胞内线粒体网络的潜在途径。
We have developed a method for transferring exogenous DNA molecules into isolated mammalian mitochondria using bacterial conjugation. In general, we accomplish this by (i) inserting an origin of DNA transfer (oriT) sequence into a DNA construct, (ii) transforming the construct into an appropriate Escherichia coli strain and then (iii) introducing the mobilizable DNA into mitochondria through conjugation. We tested this approach by transferring plasmid DNA containing a T7 promoter sequence into mitochondria that we had engineered to contain T7 RNA polymerase. After conjugation between E.coli and mitochondria, we detected robust levels of T7 transcription from the DNA constructs that had been transferred into the mitochondria. This approach for engineering DNA constructs in vitro and subsequent transfer into mitochondria by conjugation offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a potential route for transforming mitochondrial networks within mammalian cells.