TRANSFECTING MAMMALIAN MITOCHONDRIA WITH EXOGENOUS DNA

描述(申请人提供)：目前还没有因线粒体DNA(MtDNA)突变而导致母系遗传病的动物模型，原因很简单：我们不知道如何将DNA导入小鼠(或任何其他哺乳动物)线粒体，也不知道如何有效地将分离的线粒体引入哺乳动物细胞。由于未知的原因，在低等真核生物中起作用的细胞器DNA转染法在哺乳动物细胞中不起作用。在我们看来，无法将外源DNA导入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取得进展的最大障碍。在这里，我们建议将DNA导入哺乳动物线粒体，并利用遗传学手段将分离的线粒体导入哺乳动物细胞。我们有两个具体目标。在目标1中，我们将通过模仿细菌之间转移DNA的过程，即接合，将外源DNA引入线粒体。在目标2中，我们将通过模仿病原菌入侵细胞的过程将分离的线粒体引入哺乳动物细胞，通过处理含有成功感染所需的两种关键细菌蛋白的线粒体来处理哺乳动物细胞：侵袭素、刺激线粒体吞噬功能和李斯特溶素，刺激吞噬的线粒体进入细胞质。

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.