TRANSFECTING MAMMALIAN MITOCHONDRIA WITH EXOGENOUS DNA

描述（由申请人提供）：没有由于线粒体DNA （mtDNA）突变引起的母亲遗传疾病的动物模型，原因很简单：我们不知道如何将DNA转染到小鼠（或任何其他哺乳动物）的线粒体中，也不知道如何将分离的线粒体有效地引入哺乳动物细胞。由于未知的原因，在低等真核生物中起作用的细胞器DNA转染方法在哺乳动物细胞中不起作用。在我们看来，无法用外源DNA转染线粒体是理解哺乳动物线粒体遗传学，特别是人类线粒体疾病的最大障碍。我们建议在这里引入DNA到哺乳动物线粒体，并引入分离线粒体到哺乳动物细胞，使用遗传手段。我们有两个具体目标。在目标1中，我们将通过模拟细菌相互之间转移DNA的过程，即通过偶联，将外源DNA引入线粒体。这可以“直接”完成，即用含有适当构建的偶联质粒的细菌感染哺乳动物细胞，或者“间接”完成，即在体外将DNA转染到分离的纯化线粒体中，然后通过“细菌入侵”将这些线粒体转移到细胞中，如Aim #2所述。在目标#2中，我们将通过模拟致病菌侵入细胞的过程，将分离的线粒体引入哺乳动物细胞，通过用含有成功感染所需的两种关键细菌蛋白的线粒体处理哺乳动物细胞：入侵蛋白，刺激线粒体的吞噬作用，李斯特菌溶素，刺激被吞噬的线粒体释放到细胞质中。

DESCRIPTION (provided by applicant): There are no animal models of maternally-inherited diseases due to mutations in mitochondrial DNA (mtDNA), for one simple reason: we do not know how to transfect DNA into mouse (or any other mammalian) mitochondria, nor, for that matter, do we know how to introduce isolated mitochondria into mammalian cells efficiently. For unknown reasons, organellar DNA transfection methods that work in lower eukaryotes have failed to work in mammalian cells. In our view, the inability to transfect mitochondria with exogenous DNA is the single greatest stumbling block to progress in understanding mammalian mitochondrial genetics in general and human mitochondrial diseases in particular. We propose here to introduce DNA into mammalian mitochondria, and to introduce isolated mitochondria into mammalian cells, using genetic means. We have two specific aims. In Aim #1, we will introduce exogenous DNA into mitochondria by mimicking the process by which bacteria transfer DNA among each other, namely, by conjugation. This will be done either "directly," by infecting mammalian cells with bacteria harboring appropriately-constructed conjugating plasmids, or "indirectly," by transfecting DNA into isolated purified mitochondria in vitro, and then transferring these mitochondria into cells by "bacterial invasion," as outlined in Aim #2. In Aim #2, we will introduce isolated mitochondria into mammalian cells by mimicking the process by which pathogenic bacteria invade cells, by treating mammalian cells with mitochondria containing the two key bacterial proteins required for successful infection: invasin, to stimulate phagocytosis of mitochondria, and listeriolysin, to stimulate release of the engulfed mitochondria into the cytosol.