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.
| Lim, Yin Mei; de Groof, Ad J C; Bhattacharjee, Mrinal K et al. (2008) Bacterial conjugation in the cytoplasm of mouse cells. Infect Immun 76:5110-9 |
